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Dissertations / Theses on the topic 'P2X7'

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Published: 4 June 2021
Last updated: 10 March 2023

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1

Boumechache, Miyyada. "Structural and functional interaction between P2X4, P2X7 and Pannexin-1." Thesis, University of Cambridge, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.608656.

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2

O'Brien-Brown, James. "Novel P2X7 Receptor Ligands." Thesis, The University of Sydney, 2019. https://hdl.handle.net/2123/21280.

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The P2X7 receptor (P2X7R) is a purinergic receptor that plays a central role in the inflammatory response. Activation of the P2X7R releases pro-inflammatory cytokines such as interleukin 1β (IL-1β), which have been shown to underlie the pathogenesis of a number of neurodegenerative disorders. Consequently, the development of a CNS penetrant P2X7R antagonist is considered a promising target for the inhibition of neurodegenerative diseases. A series of P2X7R antagonists were synthesised to investigate which structural features of the hydrophobic moiety dictated binding site selectivity (orthosteric vs allosteric), and potency data are available for derivatives synthesised; assays to assess binding site selectivity have not currently been undertaken. To assist future pharmacological analyses, fluorescent probes based on lead compounds from the aryl cyanoguanidine and adamantyl benzamide P2X7R antagonist series were synthesised, and antagonist potency and binding affinity data for a number of derivatives are reported. Based on the original structure-activity relationship (SAR) study of the adamantyl cyanoguanidine series, a range of heterobicyclic adamantyl cyanoguanidine analogues were synthesised in order to refine the pharmacophore for potent P2X7R antagonism. The adamantyl indazoles 302 and 303 (IC50 = 18.6 ± 0.5 nM and 22.2 ± 6 nM respectively) were equipotent to the lead 19, and SAR data from this series has identified several structural requirements for potent P2X7R antagonism. Attempts to develop radioligands for visualising P2X7R expression in vivo are reported. The trifluorinated adamantyl benzamide [11C]SMW139 was progressed into first-in-human studies as a radiodiagnostic probe for identifying active areas of neuroinflammation in patients with relapsing-remitting multiple sclerosis (RRMS), with data from the small cohort suggesting it did so successfully. Further studies in larger cohorts are currently in progress.
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3

FURINI, Federica. "P2X7 receptor (P2X7R) in Systemic Lupus Erythematosus (SLE). Exploring a novel pathogenetic pathway." Doctoral thesis, Università degli studi di Ferrara, 2019. http://hdl.handle.net/11392/2487988.

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Introduzione. P2X7R è un recettore extracellulare ATP-dipendente coinvolto in processi infiammatori e autoimmuni che agiscono principalmente attraverso l'attivazione dell'inflammasoma NLRP3 e il rilascio di IL-1β, ed anche tramite processi implicati nella regolazione della proliferazione dei linfociti e nell'apoptosi cellulare. Diverse osservazioni da modelli animali e studi su paziente evidenziano un possibile collegamento tra l'asse P2X7R-NLRP3 e la patogenesi del Lupus Eritematoso Sistemico (SLE). L'asse inflammasoma-P2X7R oltre alla produzione diretta di IL-1b e IL-18, è coinvolto indirettamente nel rilascio di altre citochine implicate nella patogenesi di SLE, come IL-6. Lo scopo di questo studio è di esplorare il ruolo di P2X7R e di NLRP3- inflammasoma nel Lupus. Metodi. Sono stati arruolati 48 pazienti con SLE, 16 con (SLE-S) e 32 senza (SLE-NS) anamnesi positiva per sierosite e 20 soggetti di controllo sani (HC) abbinati per sesso ed età. Sono stati raccolti dati demografici, clinici, terapeutici e misure di outcome. I livelli plasmatici di IL-1β e IL-6 sono stati valutati mediante ELISA. Le cellule mononucleate del sangue periferico (PBMC) sono state isolate dal sangue venoso mediante sedimentazione a gradiente di Ficoll e impiegate come segue: 1) valutazione dell'espressione di P2X7R e NLRP3 mediante RT-PCR; 2) determinazione dell'attività P2X7R come incrementi dei livelli di Calcio intracellulare [Ca2 +]i indotti da Benzoyl ATP (BzATP) usando la sonda fluorescente Fura2-AM; 3) isolamento di monociti / macrofagi e valutazione del rilascio in vitro di IL-1β e IL-6 dopo stimolazione con lipopolisaccaride (LPS) e BzATP, separatamente o in combinazione. Risultati. I livelli plasmatici di IL-1β non sono risultati significativamente differenti nei pazienti con SLE rispetto a HC mentre i livelli di IL-6 sono risultati più elevati in SLE rispetto a HC, in modo significativo nei pazienti con storia di sierosite. Monociti / macrofagi isolati da pazienti affetti da SLE rilasciavano quantità inferiori di IL-1β dopo stimolazione con BzATP, mentre il rilascio di IL-6 è risultato significativamente aumentato in SLE-NS rispetto a entrambi i soggetti HC e SLE-S dopo tutti i tipi di stimolazione. L'aumento di [Ca2 +]i dopo stimolazione con BzATP era significativamente più basso nei PBMC di pazienti con SLE rispetto a PBMC da HC. La RT-PCR ha mostrato una riduzione significativa del P2X7R e un'espressione NLRP3 significativamente aumentata nei pazienti rispetto a HC. Conclusioni. I nostri dati indicano una ridotta espressione e funzione di P2X7R nei pazienti affetti da SLE rispetto ai soggetti HC e, al contrario, aumento della segnalazione di IL-6. Le possibili conseguenze della riduzione del P2X7R, principalmente sulla regolazione del network citochinico e sulla proliferazione dei linfociti, dovranno essere ulteriormente approfondite così come il ruolo dell'IL-6 come possibile obiettivo terapeutico, specialmente nei paziente con storia di sierosite.
Introduction. P2X7R is an extracellular ATP-gated receptor involved in inflammatory and autoimmune processes mainly acting through NLPR3-inflammasome activation and IL-1β release, also implicated in lymphocyte proliferation and cellular apoptosis. Several observations from animal models and patient’s studies highlight a possible link between P2X7R-NLRP3 axis and Systemic Lupus Erythematosus (SLE) pathogenesis. The P2X7R-inflammasome axis in addition to the direct production of IL-1 and IL-18, indirectly mediates the release of other cytokines implicated in the pathogenesis of SLE, such as IL-6. The aim of this study was to investigate the role of P2X7R and NLRP3-inflammasome in SLE. Methods. 48 SLE patients, 16 with (SLE-S) and 32 without (SLE-NS) history of serositis, and 20 healthy control (HC) subjects matched for sex and age were enrolled. Demographic, clinical, therapeutic data and outcome measures were collected. IL-1β and IL-6 plasma levels were evaluated by ELISA. Peripheral blood mononuclear cells (PBMCs) were isolated from venous blood by Ficoll gradient sedimentation and employed as follows: 1) evaluation of P2X7R and NLRP3 expression by RT-PCR; 2) determination of P2X7R activity as Benzoyl ATP (BzATP)-induced [Ca2+]i increments using Fura2-AM fluorescent probe; 3) isolation of monocytes/macrophages and assessment of in vitro IL-1β and IL-6 release following stimulation with lipopolysaccharide (LPS) and BzATP, either separately or in combination. Results. Plasma IL-1β levels were unmodified in SLE subjects respect to HC whereas IL-6 levels were higher in SLE than in HC, resulting significantly increased in SLE-S. Monocytes/macrophages isolated from SLE patients released lower quantities of IL-1β after stimulation with BzATP, whereas IL-6 release was significantly augmented in SLE-NS respect to both HC subjects and SLE-S after all types of stimulation. The [Ca2+]i increase following BzATP stimulation was significantly lower in PBMCs from SLE patients than in PBMCs from HC. RT-PCR showed significantly reduced P2X7R and significantly augmented NLRP3 expression in SLE patients. Conclusion. Our data indicate reduced P2X7R expression and function in SLE patients compared with HC subjects and, conversely, increased IL-6 signaling. The possible consequences of reduced P2X7R, mainly on cytokines network deregulation and lymphocyte proliferation, will be further investigated as well as the role of IL-6 as a possible therapeutic target especially in lupus serositis.
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4

Kunert, Christin. "Funktioneller Nachweis des purinergen Rezeptors P2X7 an den neuralen Progenitorzellen der murinen Subventrikularzone." Doctoral thesis, Universitätsbibliothek Leipzig, 2014. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-130838.

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Neurodegenerative Erkrankungen sind wegen steigender Prävalenz ein zunehmendes Problem in Industrieländern. In diversen Studien wurde bereits ein Zusammenhang zwischen neurodegenerativen Vorgängen und purinerger Signalübertragung aufgezeigt. Insbesondere die Rezeptoruntereinheit P2X7R ist durch seine apoptotische Wirkung bei verschiedenen Krankheiten involviert. Im Rahmen dieser Arbeit wurde die funktionelle Präsenz des P2X7R an neuralen Progenitorzellen untersucht, die von der Subventrikularzone (SVZ) der Maus isoliert wurden. Mittels Calcium-Imaging wurde die intrazelluläre Ca2+-Konzentration ([Ca2+]i) erfasst. Der P2X7R-Agonist BzATP führte in einem Mg2+-freiem Milieu zu einer deutlichen [Ca2+]i-Steigerung. Selektive (A438079, BBG) und unselektive (PPADS) Antagonisten des P2X7R sowie unterschiedliche Kationen (Zn2+, H+) inhibierten den agonistischen [Ca2+]i-Anstieg. Desweiteren bewirkte Ivermectin (IVM), ein allosterischer Modulator sowohl von P2X4R als auch von P2X7R, eine signifikante Wirksteigerung des niedrigdosierten BzATP. Dieser Effekt war an Progenitorzellen, welche P2X7R-defizienten (P2X7-/-R) Mäusen entnommen waren, nicht nachzuweisen. Weitere purinerge Antagonisten (NF449, TNP-ATP) hatten keine signifikante Wirkung an den Zellen der Wildtyp-Maus. Ebenso war der P2X1-3R-Agonist α,β-meATP wirkungslos. Ein extrazelluläres Ca2+- freies Milieu wurde zur Untersuchung der Zellen auf P2Y-R genutzt und führte zum fast vollständigen Verschwinden des agonistischen Effektes an den murinen Zellen. Allerdings zeigten P2X7-/-R-Zellen nach Entfernen von Ca2+ aus der extrazellulären Flüssigkeit eine deutliche Wirkung von BzATP, welches auf Aktivität von P2Y-R hindeutet. Zusammenfassend konnte somit durch Applikation von Agonisten, Antagonisten und Modulatoren eine Aktivität des P2X7R an den murinen Progenitorzellen der SVZ gezeigt werden, welcher möglicherweise zur Regulation der Zellproliferation beiträgt. Weitere purinerge Rezeptoren (P2X1-4R, P2Y-R) waren an den Vorläuferzellen der Wildtyp-Maus nicht sicher nachweisbar, während an murinen P2X7R-/--Zellen Aktivität von P2Y-R zu erkennen war.
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5

Weinhold, Karina. "Molekulare und biochemische Charakterisierung der purinergen Rezeptoren P2X4 und P2X7 im Alveolarepithel der Lunge." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2010. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-62141.

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Gegenstand der vorliegenden Arbeit sind die purinergen Rezeptoren P2X4R und P2X7R. Die P2XR werden durch ATP aktiviert und stellen unselektive Kationenkanäle dar, die auch für Ca2+ durchlässig sind. Beiden P2XR-Subtypen werden in den Alveolarepithel Typ I (AT I)-Zellen der Lunge exprimiert und aufgrund ihrer Kanalaktivitäten in Zusammenhang mit der alveolären Flüssigkeitshomöostase gebracht. Bei bisherigen Untersuchungen wurde jedoch die mögliche Assoziation und Modulation der P2XR durch Mikrodomänen der Zellmembran außer Acht gelassen. Ein Modell von Garcia-Marcos zeigt, dass P2X7R in Zellen der Glandula submandibularis zum Teil mit Mikrodomänen assoziiert ist. Die funktionellen Eigenschaften von P2X7R sind dabei von der Lokalisation in der Zellmembran abhängig (Garcia-Marcos et al., 2006). Die Caveolen sind eine spezielle Form von Mikrodomänen, die in der Zellmembran der AT I-Zellen auftreten. Das Hauptstrukturprotein der Caveolen im Lungenepithel ist Caveolin-1 (Cav-1). Über die Verteilung von P2X4R und P2X7R in den AT I-Zellen war bislang sehr wenig bekannt. Unsere Arbeitsgruppe identifizierte bei einer Sequenzanalyse potentielle Cav-1-Bindemotive in der Aminosäureabfolge beider P2XR (Couet et al., 1997). Die Assoziation mit den Caveolen würde die P2XR in die räumliche Nähe verschiedener Signalmoleküle bringen und die Beteiligung an downstream Events ermöglichen. Für die folgenden Analysen wurde die Alveolarepithelzelllinie E10 genutzt, da die E10-Zellen AT I-typische Eigenschaften besitzen und P2X4R, P2X7R sowie die Caveoline Cav-1 und Cav-2 aufweisen. Die Untersuchungen konzentrierten sich auf die Assoziation von P2X4R und P2X7R mit Mikrodomänen der Zellmembran sowie die wechselseitige Beziehung der P2XR. Besonders wurde dabei auf die Assoziation der P2XR mit Cav-1 eingegangen. Zusätzlich wurde in vitro die Interaktion der C-terminalen Bereiche der beiden P2XR mit Membranlipiden untersucht. Einige Membranlipide sind eng mit weiteren Signalmolekülen verknüpft. Aus diesem Grund wurde die Auswirkungen der Reduzierung von P2X4R und P2X7R auf den Proteingehalt der Ca2+-aktivierbaren downstream-Effektoren PKCβI und CaM analysiert. Die Auswertungen der Ergebnisse ergaben Folgendes: P2X4R und P2X7R sind Subtyp-spezifisch in den Mikrodomänen der Zellmembran von E10-Zellen verteilt. Mit Hilfe von biochemischen und immunfluoreszenz-mikroskopischen Methoden konnte die Assoziation von P2X4R und P2X7R mit Mikrodomänen nachgewiesen werden. P2X7R ist zum Teil mit Cav-1 assoziiert, wobei Förster Resonanz Energie Transfer (FRET)-Analysen ergaben, dass beide Proteine partiell einen Abstand von kleiner als 10 nm zueinander aufweisen. Durch die Subtyp-spezifische Verteilung könnte die Funktionalität der P2XR-Subtypen spezifisch durch die Bestandteile der Mikrodomänen moduliert und reguliert werden (Martens et al., 2001). P2X4R und P2X7R sind in hochmolekularen Proteinkomplexen assoziiert. Die Ausbildung von hochmolekularen Proteinkomplexen wird in Zusammenhang mit der Assoziation von Proteinen mit Mikrodomänen diskutiert (Zurzolo et al., 2003). Die Untersuchung der molekularen Organisation von P2X4R und P2X7R in E10-Zellen mittels blue native- und high resolution clear native-PAGE zeigte, dass beide P2XR mit hochmolekularen Proteinkomplexen assoziiert sind. P2X7R konnte in drei Komplexen nachgewiesen werden. Im ersten Komplex von ~760 kDa liegt P2X7R mit Cav-1 assoziiert vor, während der dominant auftretende, zweite P2X7R-Subkomplex von ~580 kDa vermutlich nicht mit dem co-migrierten Cav-1/Cav-2-Komplex in Verbindung steht. Der dritte P2X7R-assoziierte Komplex war zusammen mit P2X4R bei ~430 kDa nachweisbar und Immunpräzipitationen bestätigten, dass P2X4R und P2X7R in einem Komplex miteinander assoziiert sind (Weinhold et al., 2010). P2X4R und P2X7R stehen in Wechselbeziehung zueinander. Diese Ergebnisse der siRNA-induzierte Herabregulation von P2X4R und P2X7R lassen vermuten, dass die beiden Rezeptoren direkt oder indirekt miteinander verbunden sind. So führte die Reduzierung von P2X4R zur Erhöhung des P2X7R-Proteingehaltes. Dabei nimmt P2X7R in der Zellmembran zu und verändert seine Verteilung nicht. Umgekehrt nimmt der Proteingehalt von P2X4R in den E10-Zellen zu, wenn P2X7R herabreguliert wird. Die Zunahme von P2X4R in der Zellmembran konnte zwar durch die Biotinylierung der Oberflächenproteine nachgewiesen werden, aber die Verteilung von P2X4R verschob sich zugunsten des intrazellulären P2X4R-Anteils. Vermutlich führt die Reduzierung von P2X7R zu Störungen im exo-/endozytotischen System. Die wechselseitige Zunahme der P2XR in den Mikrodomänen weist zudem auf einen kompensatorischen Mechanismus hin. Negativ geladene Phospholipide interagieren direkt mit den C-terminalen Abschnitten der P2XR. Mit den in vitro Bindetests konnte gezeigt werden, dass die C-terminalen Enden von P2X4R und P2X7R direkt mit den negativ geladenen Phosphoinositiden PI(4)P, PI(4,5)P2, PI(3,4,5)P3 sowie mit Phosphatidsäure, Phosphatidylserin, Phosphatidylglycerol, Cardiolipin und 3 Sulfogalactosylceramid interagieren können. Die Regulation der P2XR durch diese Phospholipide, vor allem PI(4,5)P2, und die Beteiligung der P2XR an Lipid-vermittelten Signalwegen in Epithelzellen, stellen einen möglichen Link zu weiteren downstream-Signalen dar. Die Reduzierung von P2X7R beeinflusst den Proteingehalt der downstream-Effektoren PKCβI und CaM. Sowohl im Lungengewebe von P2rx7(-/-) Mäusen als auch nach der Reduzierung von P2X7R in den E10-Zellen zeigte sich, dass der Proteingehalt der Signalmoleküle PKCβI und CaM vermindert war. Reduzierung von P2X4R hatte dagegen kaum Einfluss auf PKCβI und führte zur Erhöhung des CaM-Proteingehaltes, vermutlich hervorgerufen durch die Zunahme von P2X7R. Beide downstream-Effektoren sind in Mikrodomänen (Caveolen) der Zellmembran lokalisiert und können sowohl durch Lipid-vermittelte Signale als auch durch einen Kanal-vermittelten Ca2+-Einstrom aktiviert und reguliert werden. Die Ergebnisse der vorliegenden Arbeit zeigten, dass P2X4R und P2X7R in AT I-Zellen der Lunge nicht nur Kanaleigenschaften besitzen, sondern durch die Assoziation mit unterschiedlichen Mikrodomänen an verschiedene Signalwege gekoppelt sind. Trotzdem ist bisher wenig über die Funktionen der P2XR in AT I-Zellen hinsichtlich der Beteiligung an apoptotischen Prozessen, der Proliferation, der Differenzierung oder Migration und Wundheilung bekannt (Barth and Kasper, 2009). Aufgrund der komplexen Funktion, vor allem durch die Assoziation mit Cav-1 und der Wechselbeziehung mit dem P2X4R, wird der P2X7R für zukünftige Forschungen im alveolären Lungenepithel von Bedeutung sein. Barth K, Kasper M (2009) Membrane compartments and purinergic signalling: occurrence and function of P2X receptors in lung. FEBS J 276:341-353. Couet J, Li S, Okamoto T, Ikezu T, Lisanti MP (1997) Identification of peptide and protein ligands for the caveolin-scaffolding domain. Implications for the interaction of caveolin with caveolae-associated proteins. J Biol Chem 272:6525-6533. Garcia-Marcos M, Perez-Andres E, Tandel S, Fontanils U, Kumps A, Kabre E, Gomez-Munoz A, Marino A, Dehaye JP, Pochet S (2006) Coupling of two pools of P2X7 receptors to distinct intracellular signaling pathways in rat submandibular gland. J Lipid Res 47:705-714. Martens JR, Sakamoto N, Sullivan SA, Grobaski TD, Tamkun MM (2001) Isoform-specific localization of voltage-gated K+ channels to distinct lipid raft populations. Targeting of Kv1.5 to caveolae. J Biol Chem 276:8409-8414. Weinhold K, Krause-Buchholz U, Rödel G, Kasper M, Barth K (2010) Interaction and interrelation of P2X7 and P2X4 receptor complexes in mouse lung epithelial cells. Cell Mol Life Sci 67:2631-2642. Zurzolo C, van Meer G, Mayor S (2003) The order of rafts. Conference on microdomains, lipid rafts and caveolae. EMBO Rep 4:1117-1121.
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Prudic, Kirsten [Verfasser]. "Charakterisierung koexprimierter humaner purinerger P2X4- und P2X7-Rezeptoren in Xenopus Laevis Oozyten / Kirsten Prudic." Halle, 2017. http://d-nb.info/1130148157/34.

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7

Stevenson, Diane J. "P2X7, inflammation and gastrointestinal disease." Thesis, University of Nottingham, 2008. http://eprints.nottingham.ac.uk/28897/.

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The inflammatory bowel diseases, ulcerative colitis and Crohn's disease are characterised by spontaneously relapsing and remitting inflammation, associated with increased mucosal levels of the inflammatory cytokine, interleukin-1 (IL-1)β. IL-1β processing and release is mediated by ATP stimulation of the purine receptor, P2X7. P2X7 is a membrane ion channel highly expressed in immune cells. Signal transduction occurs via rapid cation exchange, plasma membrane depolarisation and increased intracellular calcium. Additionally, prolonged or repeated P2X7 stimulation leads to formation of a non-selective membrane pore permeable to small molecules, and ultimately to cell death. The aim of this project was to investigate the properties of the P2X7 receptor in mononuclear cells, to show that it is associated with IL-1β release in the colon, and that this release can be modified by P2X7 antagonists. Studies of ethidium bromide uptake, a functional assay, showed that P2X7 receptors are present on LPMCs and displayed properties similar to those of PBMCs and THP-1 cells. P2X7 receptor-stimulation released mature IL-1β from LPMCs in a dose-dependent manner that, in IBD patients, matched the severity of their inflammation, and could be markedly reduced by P2X7 antagonists. P2X7 stimulation also results in increased exposure of phosphatidylserine on the outer cell membrane (PS flip), often considered to be a marker of apoptotic cell death. P2X7-stimulated PS flip however is reversible and is not associated with cell death following brief stimulation times. Cell death caused by longer stimulation did not have features of apoptosis, was more evident in monocytes than lymphocytes, with LPMCs being less susceptible than PBMCs and THP-1 cells. These studies have shown that the P2X7 receptor is intimately involved in the release of IL-1β from human colonic mononuclear cells, that the release is greater in cells from IBD tissue and can be markedly inhibited by P2X7 antagonists.
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Hempel, Christoph. "Neue Modulatoren des P2X7-Rezeptors." Doctoral thesis, Universitätsbibliothek Leipzig, 2015. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-161341.

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P2X7-Rezeptoren stellen Schlüsselmoleküle bei der Entstehung und Aufrechterhaltung proinflammatorischer Zustände, chronischer Schmerzen sowie der neuroglialen Kommunikation dar. Ihre Aktivität wird durch eine Vielzahl zellbiologischer Mechanismen beeinflusst. Dazu gehört die allosterische Modulation durch extrazelluläre niedermolekulare Stoffe. Die Entwicklung selektiver und potenter P2X7-Modulatoren ist darum Gegenstand intensiver Forschung. Bisher sind jedoch keine Pharmaka für die klinische Anwendung verfügbar. Die Untersuchung zugelassener pharmakologischer Substanzen in einem akademischen Screening erbrachte eine hohe Trefferrate für P2X7-Rezeptoren. In dieser Arbeit wird die P2X7-Wirkung einiger der potentesten allosterischen Modulatoren genauer charakterisiert. Das Antihistaminikum Clemastin stellt dabei einen positiven allosterischen Modulator dar, der den Rezeptor gegenüber niedrigeren ATP-Konzentrationen sensibilisiert. Ivermectin, ein häufig angewendetes Anthelminthikum, konnte als potenzierender Modulator des humanen P2X7-Rezeptors charakterisiert werden. Mit den Phenothiazinen Prochlorperazin und Trifluoperazin zeigen sich schließlich ZNS-gängige Inhibitoren der ATP-induzierten P2X7-Aktivität, die für weiterführende in vivo-Untersuchungen hilfreiche pharmakologische Werkzeuge darstellen können.
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AMOROSO, Francesca Saveria. "P2X7 Receptor: Warburg effect revisited." Doctoral thesis, Università degli studi di Ferrara, 2012. http://hdl.handle.net/11392/2389273.

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Ability to adapt to conditions of limited nutrient supply is a key feature of all cells. This may require a complex re-organization of metabolic pathways to balance energy generation and production of biosynthetic intermediates. Several fast-growing cells overexpress the P2X7 receptor (P2X7R) for extracellular ATP. A peculiar feature of this receptor is that it allows growth in the absence of serum. We show here that transfection of P2X7R allows proliferation of HEK293 (HEK293-P2X7) cells not only in the absence of serum but also in low (4 mM) glucose and strongly increases lactate output compared to mock-transfected cells (HEK293-mock). In HEK293-P2X7 lactate output is further stimulated upon addition of exogenous ATP or of the mitochondrial uncoupler FCCP. In another tumour cell line constitutively expressing the P2X7R, the human neuroblastoma cell line ACN, lactate output is also dependent on P2X7R function. P2X7R-expressing cells up-regulate a) the glucose transporter Glut-1, b) the glycolytic enzymes glyceraldehyde 3-phosphate dehydrogenase (G3PDH), c) pyruvate kinase M2 (PK-M2) and d) pyruvate dehydrogenase kinase 1 (PDHK1), e) increase phosphorylated Akt/PKB (ph- Akt/PKB) and f) the level of intracellular glycogen stores. In HEK293-P2X7 cells glucose deprivation strongly increases lactate production, expression of glycolytic enzymes and ph-Akt/PKB level. These data show that the P2X7R has an intrinsic ability to reprogram cell metabolism to meet the needs imposed by adverse environmental conditions.
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SARTI, Alba Clara. "P2X7 expression modulates mitochondrial metabolism." Doctoral thesis, Università degli studi di Ferrara, 2016. http://hdl.handle.net/11392/2403380.

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L’espessione del recettore P2X7 modula il metabolismo mitocondriale. Il recettore P2X7 è principalmente conosciuto per la sua abilità nel causare morte cellular dovuta ad una prolunata attivazione data dall’ATP, tramite un aumento della permeabilizzazione della membrane plasmatica. Al contrario una brave attivazione causa una modificazioni della concentrazione intracellulare di cationi che si associa a differenti processi fisiologici come induzione della cascata infiammatoria, proliferazione e soppravivemza cellulare. Negli ultimi anni si è cercato di comprendere meglio i meccanismi tramite i quali il recettore P2X7 supporta il metabolismo energetico delle cellule. Il nostro laboratorio ha in precedenza dimostrato come il recettore P2X7 ha un effetto trofico sul metabolismo energetico cellulare tramite l’aumento del potenziale mitocondriale di membrane e la sintesi di ATP. Al contrario stimolazione farmacologica del recettore purinergico causa frammentazione mitocondriale e collasso del potenziale di membrane mitocondriale. Questi dati portano in luce l’importante ruolo del P2X7 nel modulare il metabolismo mitocondriale. Nel presente studio dimostraimo come il recettore P2X7 è presente a livello dei mitocondri e in seguito a attivazione si abbia un suo aumento in questi siti. Inoltre delezione genetica del recettore P2X7 compromette la respirazione mitocondriale, il potenziale di membrane e l’abilita di produrre ROS. Questo stato cellulare de-energizzato provoca un impatto negativo sulle diverse funzioni cellulari come la migrazione. Queste osservazioni dimostrano come il P2X7 gioca un ruolo centrale nell’omeostasi energetica cellulare e nei processi che la coinvolgono.
P2X7 expression modulates mitochondrial metabolism. The P2X7 receptor is a trimeric ATP-gated cation channel best known for its ability to cause plasma membrane permeabilization and cell death after prolonged exposure to extracellular ATP. However, recent data show that its brief activation triggers rapid inward cation currents and intracellular signalling pathways associated with a multiplicity of physiological processes such as induction of the inflammatory cascade, cell proliferation and survival. Recently, there has been an increased effort to understand the mechanism by which P2X7 supports energy-requiring cell functions. We previously showed that basal P2X7 expression has a trophic effect on cellular energetics as it increases mitochondrial potential and ATP synthesis, while on the contrary pharmacological P2X7 stimulation causes mitochondrial potential collapse and fragmentation. These findings point to major role for P2X7 in the modulation of mitochondrial metabolism. In the present study we show that P2X7 localizes to the mitochondria especially following activation. Furthermore P2X7 genetic deletion severely impairs mitochondrial respiration, mitochondrial membrane potential and ability to produce ROS. Decreased energy generation impacts negatively on key cell functions such as migration. These observations demonstrate the central role played by P2X7 in the modulation of cellular energy homeostasis and energy-requiring processes.
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11

Taylor, Simon Richard James. "The P2X7 receptor in immune cells." Thesis, Imperial College London, 2009. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.508719.

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The P2X7 receptor is a cation channel activated by high concentrations of ATP. Its stimulation is pro-inflammatory, with activation resulting in the release of cytokines (notably IL-1β), changes in plasma membrane lipid distribution, and cell death. A central role for P2X7 in IL-1β secretion via the NALP3 inflammasome has been confirmed in gene-deficient mice generated by GlaxoSmithKline (GSK) and Pfizer. It is abundantly expressed on cells of the immune system and may play a role in the pathogenesis of autoimmune disease, notable systemic lupus erythematosus (SLE). Indeed, P2X7 has become an important potential therapeutic target, and antagonists are currently in Phase II clinical trials for treatment of rheumatoid arthritis and chronic obstructive pulmonary disease. In this thesis, I describe my investigation into the role of the P2X7 receptor in immune function, examining in detail the responses of murine T cells, macrophages and dendritic cells to P2X7 stimulation. Investigation of T cell responses reveals a novel form of cell death in which cells initially shrink and then swell, before undergoing catastrophic lysis with release of cellular contents into the surrounding milieu, a process which I have termed aponecrosis as it bears features of both apoptosis and necrosis. In addition, I report a detailed characterisation of immune cells from the GSK P2X7-/- mouse. Functional and mRNA data demonstrate tissue-specific 'leakiness', such that P2X7 expression is ablated in P2X7-/- macrophages, but not in P2X7-/- T cells. This explains previous paradoxical experimental and immunohistochemical data achieved with P2X7-/- mice without the need to invoke the expression of an additional P2X7-like protein cross-reactive with P2X7 antibodies. Finally, I report the use of a mouse model of antibody-mediated glomerulonephritis to demonstrate that P2X7 plays a key pro-inflammatory role in immune-mediated injury and that this receptor is a possible therapeutic target in vivo.
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12

Gu, Baijun. "THE P2X7 RECEPTOR OF HUMAN LEUKOCYTES." Thesis, The University of Sydney, 2003. http://hdl.handle.net/2123/501.

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Lymphocytes from normal subjects and patients with B-chronic lymphocytic leukemia (B-CLL) show functional responses to extracellular ATP characteristic of the P2X7 receptor. These responses include opening of a cation selective channel/pore which allows entry of the fluorescent dye, ethidium+ and activation of a membrane metalloprotease which sheds the adhesion molecule L-selectin. In this thesis, the surface expression of P2X7 receptors was measured in normal leucocytes, platelets and B-CLL lymphocytes and compared with their functional responses. Monocytes showed 4-5 fold greater expression of P2X7 than B-, T- and NK- lymphocytes, while P2X7 expression on neutrophils and platelets was weak. All cell types demonstrated abundant intracellular expression of this receptor. All 12 subjects with B-CLL expressed surface P2X7 at about the same level as for B-lymphocytes from normal subjects. P2X7 function, measured by ATP-induced uptake of ethidium, correlated closely with surface expression of this receptor in normal and B-CLL lymphocytes and monocytes. However, the ATP-induced uptake of ethidium into the malignant B-lymphocytes in 3 patients was low or absent. The lack of P2X7 function in these B-lymphocytes was confirmed by the failure of ATP to induce Ba2+ uptake into their lymphocytes. This lack of function of the P2X7 receptor resulted in a failure of ATP-induced shedding of L-selectin, an adhesion molecule which directs the recirculation of lymphocytes from blood into the lymph node. To study a possible genetic basis of non-functional P2X7 receptor, we sequenced DNA coding for the carboxyl terminal tail of P2X7. In 33 of 130 normal subjects a heterozygous nucleotide substitution (1513A--C) was found while 3 subject carried the homozygous substitution which codes for glutamic acid to alanine at amino acid position 496. Surface expression of P2X7 on lymphocytes was not affected by this 496Glu--Ala polymorphism demonstrated both by confocal microscopy and immunofluorescent staining. Monocytes and lymphocytes from the 496Glu--Ala homozygote subject expressed non-functional receptor while heterozygotes showed P2X7 function which was half that of wild type P2X7. Results of transfection experiments showed the mutant P2X7 receptor was non-functional when expressed at low receptor density but regained function at a high receptor density. This density-dependence of mutant P2X7 function was also seen on differentiation of fresh monocytes to macrophages with interferon-gamma which upregulated mutant P2X7 and partially restored its function. P2X7-mediated apoptosis of lymphocytes was impaired in homozygous mutant P2X7 compared with wild type. The data suggest that the glutamic acid at position 496 is required for optimal assembly of the P2X7 receptor. Apart from the 496Glu--Ala polymorphism, three other single nucleotide polymorphisms, 155His--Tyr, 348Ala--Thr and 568Ile--Asn were also found in the P2X7 receptor. The site directed mutant cDNA were generated for all 3 polymorphisms and transfected into HEK293 cells to study the impact of these polymorphisms on P2X7 function. Results suggested that Ile568 is important for P2X7 protein trafficking to cell surface. Further study of these two loss-of-function polymorphisms (496Glu--Ala and 568Ile--Asn) may help better understanding of the functional domains in the P2X7 receptor and its role in CLL, lymphoma and infectious diseases. Conclusions: 1.P2X7 receptor is expressed in human leukocytes, including lymphocytes, natural killer cells as well as monocytes, on both surface and intracellular locations. 2.Both the expression and function of P2X7 are highly variable between in human individuals. Non-functional P2X7 receptors are found in some subjects, including both normal subjects and CLL patients, and are often associated with defects in ATP-induced cytotoxicity and L-selectin shedding. 3.Two single nucleotide polymorphisms (SNPs), 496Glu--Ala and 568Ile--Asn, are found at low frequency in the human population and lead to the loss-of-function of P2X7. Both permeabllity function and the downstream effects mediated by P2X7 are affected by these two SNPs. The mechanisms for the loss-of-function differs between the two polymorphisms.
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13

Gu, Baijun. "THE P2X7 RECEPTOR OF HUMAN LEUKOCYTES." University of Sydney. Medicine, 2003. http://hdl.handle.net/2123/501.

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Lymphocytes from normal subjects and patients with B-chronic lymphocytic leukemia (B-CLL) show functional responses to extracellular ATP characteristic of the P2X7 receptor. These responses include opening of a cation selective channel/pore which allows entry of the fluorescent dye, ethidium+ and activation of a membrane metalloprotease which sheds the adhesion molecule L-selectin. In this thesis, the surface expression of P2X7 receptors was measured in normal leucocytes, platelets and B-CLL lymphocytes and compared with their functional responses. Monocytes showed 4-5 fold greater expression of P2X7 than B-, T- and NK- lymphocytes, while P2X7 expression on neutrophils and platelets was weak. All cell types demonstrated abundant intracellular expression of this receptor. All 12 subjects with B-CLL expressed surface P2X7 at about the same level as for B-lymphocytes from normal subjects. P2X7 function, measured by ATP-induced uptake of ethidium, correlated closely with surface expression of this receptor in normal and B-CLL lymphocytes and monocytes. However, the ATP-induced uptake of ethidium into the malignant B-lymphocytes in 3 patients was low or absent. The lack of P2X7 function in these B-lymphocytes was confirmed by the failure of ATP to induce Ba2+ uptake into their lymphocytes. This lack of function of the P2X7 receptor resulted in a failure of ATP-induced shedding of L-selectin, an adhesion molecule which directs the recirculation of lymphocytes from blood into the lymph node. To study a possible genetic basis of non-functional P2X7 receptor, we sequenced DNA coding for the carboxyl terminal tail of P2X7. In 33 of 130 normal subjects a heterozygous nucleotide substitution (1513A--C) was found while 3 subject carried the homozygous substitution which codes for glutamic acid to alanine at amino acid position 496. Surface expression of P2X7 on lymphocytes was not affected by this 496Glu--Ala polymorphism demonstrated both by confocal microscopy and immunofluorescent staining. Monocytes and lymphocytes from the 496Glu--Ala homozygote subject expressed non-functional receptor while heterozygotes showed P2X7 function which was half that of wild type P2X7. Results of transfection experiments showed the mutant P2X7 receptor was non-functional when expressed at low receptor density but regained function at a high receptor density. This density-dependence of mutant P2X7 function was also seen on differentiation of fresh monocytes to macrophages with interferon-gamma which upregulated mutant P2X7 and partially restored its function. P2X7-mediated apoptosis of lymphocytes was impaired in homozygous mutant P2X7 compared with wild type. The data suggest that the glutamic acid at position 496 is required for optimal assembly of the P2X7 receptor. Apart from the 496Glu--Ala polymorphism, three other single nucleotide polymorphisms, 155His--Tyr, 348Ala--Thr and 568Ile--Asn were also found in the P2X7 receptor. The site directed mutant cDNA were generated for all 3 polymorphisms and transfected into HEK293 cells to study the impact of these polymorphisms on P2X7 function. Results suggested that Ile568 is important for P2X7 protein trafficking to cell surface. Further study of these two loss-of-function polymorphisms (496Glu--Ala and 568Ile--Asn) may help better understanding of the functional domains in the P2X7 receptor and its role in CLL, lymphoma and infectious diseases. Conclusions: 1.P2X7 receptor is expressed in human leukocytes, including lymphocytes, natural killer cells as well as monocytes, on both surface and intracellular locations. 2.Both the expression and function of P2X7 are highly variable between in human individuals. Non-functional P2X7 receptors are found in some subjects, including both normal subjects and CLL patients, and are often associated with defects in ATP-induced cytotoxicity and L-selectin shedding. 3.Two single nucleotide polymorphisms (SNPs), 496Glu--Ala and 568Ile--Asn, are found at low frequency in the human population and lead to the loss-of-function of P2X7. Both permeabllity function and the downstream effects mediated by P2X7 are affected by these two SNPs. The mechanisms for the loss-of-function differs between the two polymorphisms.
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14

Girotti, Priscila Azevedo. "Análise morfoquantitativa dos neurônios mioentéricos e submucosos imunorreativos aos receptores P2X2 e P2X7, ao óxido nítrico sintase (NOS), à calretinina, à calbindina e à colina acetil transferase (ChAT) do colo distal de ratos submetidos à desnutrição e à renutrição protéica." Universidade de São Paulo, 2008. http://www.teses.usp.br/teses/disponiveis/42/42131/tde-09102008-125826/.

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Este projeto, analisou a distribuição dos neurônios nos plexos mioentérico (PM) e submucoso (PS) imunorreativos aos receptores P2X2 (ir) e P2X7 (ir), calbindina (Calb-ir), calretinina (Calr-ir), colina acetil transferase (ChAT-ir) e ao óxido nítrico sintase (NOS-ir) do colo distal de ratos submetidos à desnutrição a renutrição protéica. Utilizaram-se colos distais de ratos nutridos (N42), desnutridos (D42) e renutridos (RN42). Os resultados do plexo PM, demonstraram que 100% dos neurônios Calb-ir, Calr-ir, ChAT-ir e NOS-ir, expressavam os receptores P2X2-ir e P2X7-ir nos três grupos. A densidade neuronal no PM, demonstrou um aumento de 20% a 97% dos neurônios receptores P2X2-7-ir, Calr-ir, ChAT-ir e NOS-ir e no PS foi de 29% a 75%, ambos D42 e recuperação no RN42. O perfil neuronal P2X7-ir, Calb-ir, Calr-ir e ChAT-ir do PM demonstrou diminuição de 28% a 40% e no PS os neurônios P2X2-7-ir, Calb-ir e ChAT-ir de 19% a 47% no D42. Concluí-se que, a desnutrição afeta os neurônios entéricos havendo recuperação na renutrição, podendo influenciar nas funções gastrintestinais.
The aim of the work was to analyze the distal colon myenteric (MN) and submucous (SN) neurons immunoreactive for P2X2-7 receptors, calbindin (Calb-ir), calretinin (Calr-ir), choline acetyltransferase (ChAT) and nitric oxide synthase (NOS) of the animals submitted to undernutrition and refeeding proteic. Distal colon was used from nourished (N42), undernourished (D42) and refeeding (RN42) rats. The results have shown 100% coexpression of the myenteric and submucous Calb-ir, Calr-ir, ChAt-ir e NOS-ir neurons with P2X2-7-ir receptors. The MN density have shown increase of the 20% and 97% of the P2X2-7-ir, Calr-ir, ChAT-ir e NOS-ir neurons of the D42 group, and the SN have been increased 29% a 75% in the D42 group. In the MN neuronal profile have shown decrease P2X7-ir, Calb-ir, Calr-ir and ChAT-ir neurons of the 28% to 40% and in the PS P2X2-7-ir, Calb-ir and ChAT-ir of the 19% a 47% neurons in the D42 group. I concluded that, the undernutrition affects the enteric neurons and there was recuperation in the refeeding, this can influence the gastrintestinal functions.
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15

Kopp, Robin [Verfasser], and Annette [Akademischer Betreuer] Nicke. "Analysis of P2X7 protein complexes in a P2X7-EGFP BAC transgenic mouse model / Robin Kopp ; Betreuer: Annette Nicke." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2020. http://d-nb.info/1231712279/34.

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16

Hillman, Katherine Anne. "P2X7 in normal and cystic kidney development." Thesis, University College London (University of London), 2004. http://discovery.ucl.ac.uk/1446590/.

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P2X7 is a unique member of the P2X family of membrane receptors for extracellular ATP. As well as being a non-selective cation channel, this membrane receptor is implicated in several biological functions, including cell death and proliferation. P2X7 expression, initially thought restricted to immune cells, also occurs in epithelial and other cell types. I hypothesised that P2X7 is functionally expressed in the renal tract, particularly in situations in which cell turnover is prominent. I have demonstrated expression of P2X7 in both mouse and human kidney. During mouse nephrogenesis P2X7 was detected in the condensing mesenchyme in early metanephrogenesis and was subsequently restricted to derivatives of the ureteric bud i.e. collecting duct and ureter. P2X7 was immunolocalised in regions of cell turnover, consistent with a role for the nucleotide receptor in nephrogenesis. P2X7 was also detected in cystic collecting ducts of both the cpk/cpk mouse model of autosomal recessive polycystic kidney disease (ARPKD) and human ARPKD. Next I investigated the potential function of P2X7 in cystogenesis using a 3D suspension culture model from the cpk/cpk mouse. Exposure to agonists of the P2X7 receptor caused a significant reduction in numbers of cysts forming in vitro. This was inhibited by P2X7 antagonists, and was greater than the response to other nucleotides, supporting a specific mediation by the P2X7 receptor. My study did not demonstrate a significant effect on markers of cell proliferation, apoptosis or necrosis correlating with P2X7-mediated reduction in cyst numbers, suggesting an alternative function for the receptor in cyst formation. To further understand the molecular mechanisms by which P2X7 mediates its functions, particularly apoptosis, I have developed an in vitro expression system. Stable transfection of a chicken lymphocytes with rat P2X7 enabled characterisation of the receptor's membrane properties, both ion fluxes and pore potential, and established a novel tool with which to further examine the mechanisms by which P2X7 mediates cell death. Further understanding of the molecular mechanisms of this unique nucleotide receptor, and its functional roles in the kidney, particularly in the setting of polycystic kidney disease may in the future lead to a novel therapeutic target for the manipulation of progression of renal injury, via its apoptotic pathways, or other as yet undefined pathways.
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17

Jackson, Alexander Rodney. "Pharmacological Evaluation of Cyanoguanidine P2X7 Receptor Antagonists." Thesis, The University of Sydney, 2017. http://hdl.handle.net/2123/17186.

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ABSTRACT BACKGROUND AND AIMS: The P2X7 receptor (P2X7R) is an ATP-gated, non-selective cation channel highly expressed on monocytes, macrophages and microglia. Prolonged activation of the P2X7R by ATP leads to cytolytic pore formation and the release of inflammatory mediators including interleukin-1β and prostaglandin E2. Accumulating evidence suggests a role for the P2X7R in neuroinflammation and thus P2X7R antagonists might be useful in diseases including chronic pain, depression and Alzheimer’s disease. Both negative allosteric modulators of the P2X7R, such as the adamantyl benzamides, and orthosteric antagonists, such as the aryl cyanoguanidines, inhibit the ATP-induced release of IL-1β from immune cells. This shared ability to inhibit IL-1β release may explain why no attempts have been made to determine the features which promote binding to the allosteric or orthosteric site. An advantage of targeting the allosteric or orthosteric site might emerge however, if a different agonist of the P2X7R is used. An antimicrobial peptide produced within the human body, LL-37, is also able to activate the P2X7R and yet LL-37 is never used in the characterisation of new series of P2X7R antagonists. The aims of this project were to characterise a novel series of P2X7R antagonists, the adamantyl cyanoguanidines, which have a hybrid structure derived from the adamantyl benzamides and aryl cyanoguanidines. Characterisation of the adamantyl cyanoguanidines should allow determination of the features which promote binding to the orthosteric or allosteric site of the P2X7R, which was one of the primary aims of this project. A second aim was to evaluate the potential of the hybrid series for further development as P2X7R antagonists by considering their potency and physicochemical P a g e | 13 properties. The final aim was to determine if there was any advantage of targeting the orthosteric or allosteric site of the P2X7R particularly with regard to inhibiting LL-37-mediated activation of the P2X7R. METHODS: The potency of adamantyl cyanoguanidines and reference P2X7R antagonists were determined in YO-PRO-1 dye uptake assays and interleukin-1β release assays to develop structure-activity relationships. A potent member of the adamantyl cyanoguanidines and several reference P2X7R antagonists were pharmacologically characterised in Schild assays, washout studies and receptor protection studies. The ability of several negative allosteric modulators and an orthosteric antagonist to inhibit LL-37-induced dye uptake was also examined. RESULTS: More compact adamantyl cyanoguanidines including those with a methylene linker between the adamantyl and cyanoguanidine groups and no linker between the cyanoguanidine group and phenyl ring were more potent than analogues with longer linkers. Ortho-substitution of the phenyl ring or substitution of the ring with 5-quinoline led to increased potency. The potency seen in the dye uptake assay was also seen in the interleukin-1β release assay. A potent member of the series 3-19 was determined to be a slowly reversible negative allosteric modulator as was 1-17 an adamantyl benzamide. The parent aryl cyanoguanidine, A-804598, was confirmed to be an orthosteric antagonist. None of the compounds were able to inhibit LL-37-induced dye uptake. DISCUSSION AND CONCLUSIONS: The determined structure-activity relationships for the adamantyl cyanoguanidines confirm their potential for further development since the series was highly amenable to modification and several potent analogues have favourable physicochemical properties including lower molecular weight. Since 3-19 P a g e | 14 was a negative allosteric modulator despite its structural similarity to A-804598 this suggests the adamantyl group promotes binding to the allosteric site and that cyanoguanidine is a tolerated bioisostere for the acetamide group at the allosteric site. The 5-quinolinyl group, in the appropriate position, facilitates binding to either site. The failure of multiple P2X7R antagonists to inhibit LL-37-induced dye uptake is concerning since LL-37 alone has been shown to induce the release of interleukin-1β from human monocytes. Future research must determine if LL-37 is responsible for cytokine release in vivo and develop small molecule antagonists of the action of LL-37.
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18

Benzaquen, Jonathan. "Étude de l’impact de l’expression de variants d’épissage sur les fonctions biologiques de P2RX7 : rôle dans l’adénocarcinome broncho-pulmonaire humain." Electronic Thesis or Diss., Université Côte d'Azur, 2020. http://www.theses.fr/2020COAZ6049.

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Le cancer du poumon, découvert classiquement à un stade avancé et non opérable, est la première cause de décès par cancer. Les avancées en immunothérapie anti tumorale ont changé le pronostic de cette pathologie en positionnant cette approche comme un élément principal de la prise en charge à ce jour. Néanmoins la survie à cinq ans du cancer du poumon, tous stades confondus, reste inférieure à 20% et il est donc nécessaire de mieux comprendre sa physiopathologie afin d’identifier de nouvelles approches thérapeutiques. Mon laboratoire d’accueil a récemment montré que le récepteur purinergique P2RX7 restreint le développement de tumeurs dans un modèle murin en orchestrant une réponse immunitaire anti tumorale. L’expression de P2RX7 a été montrée dans l’adénocarcinome bronchique humain (LUAD) mais sa fonction n’a jamais été étudiée. Les objectifs de mon projet de recherche étaient de comprendre pourquoi les cellules tumorales du LUAD expriment P2RX7 dont l’une des fonctions est de déclencher leur propre mort. Nous avons émis l’hypothèse que l’expression de variants d’épissage pourrait impacter les fonctions biologiques de P2RX7. J’ai montré que l’isoforme P2RX7A pleine taille et trois variants d'épissage alternatif P2RX7B, H, J sont exprimés dans les tissus LUAD et qu’il y a globalement moins de messagers dans les tissus tumoraux que dans les tissus sains adjacents. Après avoir développé un essai pour mesurer l’activité biologique de P2RX7, nous avons démontré que seul P2RX7 exprimé dans les cellules immunitaires pulmonaires est fonctionnel, et qu’il existe une altération de ses fonctions biologiques uniquement dans le compartiment tumoral. J’ai ensuite caractérisé le mécanisme moléculaire qui conduit à l’expression d’un récepteur dont la fonction biologique est altérée. Ce mécanisme repose sur l’expression différentielle du variant d'épissage P2RX7B et à la capacité de l’isoforme P2RX7B d’hétéro-oligomériser avec P2RX7A pour former un récepteur moins fonctionnel. Enfin, j’ai observé que les tumeurs des patients LUAD qui présentent un niveau d’expression élevé de P2RX7B sont très faiblement infiltrées en cellules immunitaires. L’ensemble de mes résultats positionnent P2RX7B comme un biomarqueur péjoratif pour les patients souffrant de LUAD, et comme un outil théranostique prometteur dans une perspective curative
Lung cancer, classically discovered at an advanced and non-operable stage, is the leading cause of cancer death. Advances in anti-tumor immunotherapy have changed the prognosis of this pathology by positioning this approach as a central element of therapy. Nevertheless, the five-year survival, all stages confused, remains less than 20% and it is therefore necessary to better understand the pathophysiology of this disease in order to identify new therapeutic approaches. My PhD host laboratory has recently shown that the purinergic receptor P2RX7 restricts tumor development in a murine model by orchestrating an anti-tumor immune response. The expression of P2RX7 has been shown in lung adenocarcinoma (LUAD) but its function has never been studied. The aims of my research project were to understand why LUAD tumor cells express P2RX7, one of the functions of which is to trigger their own death. We hypothesized that the expression of splice variants could impact the biological functions of P2RX7. I showed that the full size P2RX7A isoform and three alternative splice variants P2RX7B, H, J are expressed in LUAD tissues and that there are globally less messenger’s RNA in tumor tissues than in adjacent healthy tissues. After developing an assay to measure the biological activity of P2RX7, we demonstrated that only P2RX7 expressed in lung immune cells is functional, and that there is an alteration of its biological activities only into tumor compartment. I then characterized the molecular mechanism that leads to the expression of a receptor whose biological function is altered. This mechanism is relying on the differential expression of the P2RX7B splice variant and the ability of the P2RX7B isoform to hetero-oligomerize with P2RX7A to form a less functional receptor. Finally, I observed that tumors of LUAD patients having a high level of P2RX7B expression are very poorly infiltrated with immune cells.All these results position P2RX7B as a pejorative biomarker for LUAD patients and as a promising theranostic tool in a curative perspective
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19

El, Ouaaliti Malika. "Study of the expression and the role of P2X4 and P2X7 receptors in polarized murine and human macrophages." Doctoral thesis, Universite Libre de Bruxelles, 2013. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/209377.

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Throughout this work, we looked at P2X coupled pathways in macrophages. We worked on three different models of macrophages in order to establish the best model to understand the role of P2X4 receptors in the inflammation. Our work also consisted of further characterizing P2X7 receptor dependent pathways in these models.

P2X4 and P2X7 receptors are ionotropic receptors which are expressed by a variety of immune cells including macrophages. Macrophages play a very important host defense function as they are major actors in the innate immune system and they can initiate the activation of the adaptive immune system. The endogenous ligand of P2X receptors is ATP for which they share very different sensitivities. P2X4 receptors are relatively sensitive to this agonist while P2X7 receptors require concentrations > 100 μM ATP to be activated.

Our study supports the expression of P2X4 and P2X7 receptors in J774.2 murine macrophages and in human macrophages. Additionally, we worked on murine peritoneal macrophages for which the existence of P2X4 and P2X7 receptor expression had previously been shown in our lab.

A wide range of different macrophage phenotypes exist. Two extremes determine an array of phenotypes which are delimited by M1 pro-inflammatory macrophages and M2 anti-inflammatory macrophages while Mφ macrophages define the center of the array. Most of the work exposed in this study was carried out on pro-inflammatory macrophages which were obtained either by priming the cells with LPS alone (Mφ + LPS) or by polarizing them with LPS in association with IFNγ (M1).

We show in this study that LPS-primed J774.2 murine macrophages are not a good model to study the role of surface P2X4 receptor in pro-inflammatory macrophages. Additionally, we support that murine peritoneal macrophages primed with LPS are a good model to understand the hypothetical role of P2X4 receptors in the inflammation. Finally, we suggest that human M1 macrophages could be as well. Next, we also confirm that J774.2 murine macrophages, murine peritoneal macrophages and human macrophage express functional P2X7 receptors. In this study, we show that P2X7 receptors are coupled to RONS formation in J774.2 murine macrophages and to AA release through PLA2 activation in peritoneal macrophages. We show that activation of J774.2 murine macrophages with high concentrations of ATP (>600 μM) stimulates ROS formation including mitochondrial superoxide anions. In addition, our work shows the importance in using different dyes and suggests that different types of ROS play different functions in P2X7 receptors downstream pathways.

Next, we show that P2X7 receptor activation is coupled to an iPLA2 activity and that the release of free fatty acids mediated by 1 mM ATP is p-ERK1/2 dependent in LPS-primed murine peritoneal macrophages.

In addition, we have evaluated the effect of hypoxia on pathways which have been reported to be coupled to P2X7 receptor activation in pro-inflammatory human macrophages. Hypoxia does not seem to modulate P2X7 receptor functionality. However, both acute and chronic hypoxia influenced P2X7 receptors downstream pro-inflammatory coupled pathways. Finally, our work has enabled us to suggest for the first time that IFNγ plays an important function in host defense mediated by human P2X7 receptor activation in a hypoxic environment.

The effect of extracellular environment and thus different macrophage phenotypes have also been evaluated throughout this work in which we looked at the effect of polarization on P2X4 and P2X7 receptor functionality. Our work shows that LPS-priming does not modulate P2X4 receptor functionality in murine macrophages. Next, through our work, we suggest that polarization of human macrophages affects P2X4 receptor functionality in human macrophages. Additionally, our work shows that LPS affects ATP-mediated RONS formation in J774.2 murine macrophages but not P2X7-mediated AA release in primary murine macrophages.

Overall, first, our work has enabled us to suggest macrophage models to use in order to study the hypothetical role of P2X4 receptor in the inflammation mediated by macrophages. Second, it has allowed us to further understand how P2X7 receptors can act as important mediators of the immune system mediated by macrophages. In addition, many interesting observations were made in this study which allows us to propose several options for future directions. To finish, our work underlines the importance of the extracellular environment in some pathways mediated by ATP in macrophages.


Doctorat en sciences pharmaceutiques
info:eu-repo/semantics/nonPublished

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20

Safya, Hanaa. "Modulation des activités du récepteur purinergique P2X7 au cours de l’activation des lymphocytes T." Thesis, Paris 11, 2014. http://www.theses.fr/2014PA11T083/document.

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L’ATP extracellulaire, à travers l’activation du récepteur P2X7, joue un rôle important dans l’immunité inné comme signal de danger responsable de l’assemblage de l’inflammasome, de la migration des cellules immunitaires et de la mort cellulaire. Bien que le rôle de la voie ATP/P2X7 dans l’immunité adaptative reste sous-estimé, il a été rapporté que le récepteur P2X7 participe aux mécanismes de signalisation impliqués dans l’activation des lymphocytes T, leur prolifération et leur différentiation. Notre laboratoire a récemment montré que les lymphocytes T effecteurs (CD4+ ou CD8+) en fin de réponse immunitaire secondaire, exprimant à la membrane la tyrosine phosphatase de membrane B220, sont totalement résistant à l’activation du récepteur P2X7 à cause d’une perte d’adressage de ce récepteur à la membrane. Le but de ce travail de thèse est d’étudier la sensibilité des lymphocytes T, à différents stades d’activation, aux activités cellulaires induites par l’ATP, notamment le clivage de la molécule de homing CD62L ou L-sélectine, l’ouverture du canal ionique, la formation du pore et l’externalisation de la PS. Mes principaux résultats montrent que les activités cellulaires dépendantes du récepteur P2X7 sont dissociées. Les lymphocytes T au stade effecteur/mémoire sont moins sensibles au clivage de la molécule CD62L que les lymphocytes T au stade naïf et récemment activé. Les lymphocytes naïfs T récemment activé en réponse immunitaire primaire sont les plus sensibles à la formation du pore. De plus, les lymphocytes T récemment activés, aussi bien en réponse immunitaire primaire que secondaire, sont les plus sensibles à l’externalisation de la PS. Enfin, dans les lymphocytes T récemment activé, les activités de pore et d’externalisation de PS, mais pas le clivage de CD62L, sont dépendantes du taux de calcium
Extracellular ATP through the receptor P2X7 (P2X7R) plays a key role in innate immunity as a danger signal that causes the activation of the inflammasome, enhancement of immune cell migration and cell death. Although the role of the ATP/P2X7R pathway in adaptative immunity remains underestimated, it has been reported that P2X7R regulates signaling events involved in T-cell activation, proliferation, and differentiation into effector lineages. Moreover, we have previously shown that effector T lymphocytes (either CD4+ or CD8+) that express the B220 isoform of CD45 at the plasma membrane at the end of the secondary immune response are totally resistant to ATP stimulation due to loss of P2X7R membrane expression. In the present study, we compared the sensitivity of T lymphocytes to cellular activities trigerred by P2X7R according to their stage of activation. Interestingly, our results showed that P2X7-dependent cellular activities are dissociated. T lymphocytes at effector/memory stage are less sensitive to CD62L shedding than naïve or recently activated T lymphocyte during primary immune response. Naive T lymphocytes recently activated during primary immune response are the most sensitive to pore formation. Furthermore, recently activated T lymphocytes at both primary and secondary immune responses are the most sensitive to PS externalization. Finally, pore formation, PS externalization but not CD62L shedding, are dependent on calcium signaling
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21

Deplano, Simona. "Role of P2X7-mediated inflammasome activation in glomerulonephritis." Thesis, Imperial College London, 2012. http://hdl.handle.net/10044/1/17794.

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Glomerulonephritis is a major cause of kidney failure and current treatment is based on nonspecific immunosuppressive therapies. The purinergic P2X7 receptor (P2X7R) is usually not detectable in renal tissue. However, previous studies have demonstrated an increased glomerular P2X7R expression in animal models of glomerulonephritis. Furthermore, P2X7R knock-out mice have been shown to be significantly protected from antibody-mediated glomerulonephritis. P2X7R activation represents a fundamental step for the activation of the NLRP3 inflammasome which leads to the processing and release of IL-1β and IL-18. The role of the inflammasome activation in glomerulonephritis is not clear yet. The work presented in this thesis describes three aspects of P2X7R activation: cytokine production, signalling cascade following ATP stimulation and inflammasome activation. My data show that macrophages from wild type mice produce higher levels of IL-1β and IL-18 compared to macrophages from P2X7 deficient mice. ATP stimulation activates several signalling pathways in macrophages. Among them, the ribosomal pathway appears to be strictly regulated by P2X7R. To investigate the role of the inflammasome activation in glomerulonephritis I have compared macrophages from the susceptible rat strain Wistar-Kyoto with macrophages from the resistant strain Lewis. WKY macrophages express higher P2X7 mRNA and protein levels, release higher levels of IL-1β and IL-18 and exhibit a greater caspase-1 activity. Similarly, WKY nephritic glomeruli show higher P2X7, IL-1β, IL-18 and caspase-1 levels compared to Lewis glomeruli. Finally, in the attempt to identify genes responsible for the inflammasome regulation, I have examined macrophages and nephritic glomeruli from congenic rats. My data seem to indicate that the susceptibility locus Crgn2 contains one or more genes that control IL-1β and IL-18 release in macrophages. Further studies are certainly required to verify the relevance of these data. The results are important in understanding the pathogenesis of glomerulonephritis and identification of new potential therapeutic targets.
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22

Leeson, Hannah Caitlin. "P2X7 Receptor Regulation of Hippocampal Neural Progenitor Cells." Thesis, Griffith University, 2017. http://hdl.handle.net/10072/373045.

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Adult hippocampal neurogenesis plays an essential role in the formation and consolidation of new memories, spatial processing and some forms of learning. Identifying the molecular mechanisms that regulate hippocampal neural progenitor cells as they proliferate, differentiate, and are selected for either survival or cell death will provide a fundamental understanding of how this neurogenic niche coordinates these activities. Here, the roles of P2X7 receptors are examined for their influence over neural progenitor cell biology, particularly cell death, proliferation, and phagocytosis of apoptotic progenitors that have undergone programmed cell death. As a purinergic cation channel, P2X7 receptors are exceptionally versatile; their primary role is as ATP-gated calcium channels, and they have notable roles in the immune system, where they regulate cytokine release and form large transmembrane pores resulting in cell death. By acting as scavenger receptors, they can also mediate phagocytosis. These diverse roles were investigated in neural progenitor cells of the adult murine hippocampal neurogenic niche. Primary cultures of hippocampal neural progenitor cells were derived from adult female C57BL/6 mice and characterised using multimarker immunocytochemistry as P2X7 receptor positive type 2 neural progenitor cells, as defined by Sox2pos, nestinpos, BLBPpos, Mash1pos/neg, vimentinpos, Pax6pos, Prox1pos, DCXneg, GFAPneg staining patterns. For some experiments, cultures derived from P2X7 knock out mice (Pfizer) were also used. Calcium influx assays using the indicator dye Fluo-8-AM demonstrated functional activity of P2X7 receptors with the general agonist ATP (1 mM) and the more specific agonist BzATP (100 μM). Ethidium bromide uptake demonstrated that P2X7 receptors were able to form large transmembrane pores, a canonical function unique to this receptor, and confirmed the presence of a full length protein, as opposed to various splice variants. Live cell confocal microscopy revealed hippocampal neural progenitors are capable of phagocytosing fluorescent latex beads, and flow cytometry in conjunction with specific inhibitors demonstrated that P2X7 receptors are capable of facilitating this phagocytosis. The effects of purinergic signalling on neural progenitor proliferation were assessed using the thymidine analogue EdU. P2X7 receptors activated with either extracellular ATP or BzATP showed a significant dose-dependent decrease in proliferation. Cell death was not observed under these conditions and proliferation could be rescued upon exchange of medium. P2X7 receptor inhibition reduced the effects of extracellular ATP on proliferation, and use of neural progenitor cultures derived from genetically null mice corroborated this observation. Convergence with growth factor signalling pathways was also explored. The data presented here provides good evidence that P2X7 receptors function as scavenger receptors in the absence of ATP, allowing neural progenitor cells to phagocytose their apoptotic peers during target-independent programmed cell death, as well as governing rates of proliferation in the presence of ATP, possibly by regulating calcium dependent downstream signalling. Effector molecules of calcium signalling pathways were investigated following P2X7 receptor activation to determine some of the downstream mechanisms involved in P2X7 receptor mediated decreases in proliferation. Live cell calcium imaging identified the instigation of secondary calcium oscillations following extracellular ATP application; it was hypothesised that the decrease in proliferation was due to calcium dependent signalling cascades, involving calcium release from internal stores. Using confocal microscopy, calcium dependent transcription factors NFκB and NFAT1 were evaluated for their potential to translocate to the nucleus following purinergic stimulation. Extracellular ATP did not cause translocation of NFκB or NFAT1. A possible convergence with growth factor signalling pathways was investigated as the growth factors present in culture conditions exert powerful regulation over the cells and also utilise calcium and endoplasmic reticulum signalling to exert their effects. Inhibition of proteins involved in endoplasmic reticulum signalling caused a decrease in proliferation, as did growth factor withdrawal. Transcription factor analysis revealed that withdrawal of both EGF and bFGF caused NFAT1, but not NFκB, to translocate to the nucleus, a novel finding in these cells. The data presented here is among the first to examine the dichotomous signalling roles of P2X7 receptors in adult hippocampal neural progenitor cells. In mature neurons, P2X7 receptors have been implicated in various pathologies, and may present a therapeutic target for a number of neurological disorders. Understanding how these receptors regulate the physiology of stem and progenitor cells is an important first step in developing any regenerative therapies. Given the crucial role neurogenesis plays in both memory formation and hippocampal function, understanding these biological mechanisms is essential to addressing significant questions regarding neurogenesis and regeneration.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Natural Sciences
Science, Environment, Engineering and Technology
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23

SCUSSEL, BERGAMIN Leticia. "Pathophysiology of the P2X7 receptor in the skeleton." Doctoral thesis, Università degli studi di Ferrara, 2021. http://hdl.handle.net/11392/2488315.

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The biological effects of ATP are mediated by purinergic receptors, and, among them, P2X7R exhibits unique molecular and functional features. P2X7R is involved in a series of skeletal physiological activities, and it has also been associated in some bone and joint diseases. Thereby, P2X7R can be considered a potential drug target. The work of this thesis is part of a broader project aimed at identifying new molecular targets for the development of innovative therapeutic strategies to be used for the treatment of pathologies that affect bone and cartilage, and for slowing the physiological degeneration process due to aging. Aligned with this goal, the attention was also to optimize in vitro experimental models for mimicking the in vivo skeletal microenvironment, in order to obtain informative results. The main goals of the study were: - the participation of P2X7R in the maturation of cells responsible for the deposition of cellular mineralized matrix. Wharton’s jelly mesenchymal stromal cells from umbilical cord (WJ-MSCs) and osteoblasts from bone fragments (hOBs) were cultured in hypoxia and normoxia conditions, and treated with P2X7R antagonists and apyrase (scavenger of ATP and ADP). The results obtained allowed us to attribute a role to P2X7R not so much in the differentiation process of stromal cells, but in the ability of hOBs to deposit mineralized matrix. Furthermore, in investigating the possible role of osteogenic transcription factors in the modulation of P2X7R expression, the data of chromatin immunoprecipitation and transfection with specific expression vectors have shown: a. the presence in the P2RX7 gene promoter of different binding sites for osteogenic transcription factors; b. the recruitment of NFATc1 factor in specific sites of the P2RX7 promoter; c. the increase in the expression of P2X7R following the over expression of NFATc1. - the participation of P2X7R in intervertebral disc homeostasis (IVD). The mechanisms that support the onset and progression of the alteration of the IVD microenvironment are still poorly understood. It is known that the extracellular ATP is able to activate the NLRP3 inflammasome with the participation of P2X7R and the release of IL-1β. This cytokine is believed to be the key event contributing to disc degeneration. In the study of this thesis 83 IVD samples with different degrees of degeneration (Pfirrmann I-V) were collected after surgery, and used for histological analysis and for primary disc cell cultures. The data obtained so far have shown that: a. the level of tissue degeneration or cellular de-differentiation correlates with the increased expression of P2X7R and NLRP3; b. the release of IL-1β is conditioned by the presence of LPS and BzATP (P2X7R agonist), indicating an involvement of the P2X7R-NLRP3 axis. In conclusion, we demonstrated that P2X7R and its downstream target (NLRP3 inflammasome) are involved in pathogenesis of IVD degeneration. Overall, the data obtained suggest that P2X7R represents a good molecular target to promote the anabolic activity of the bone cells responsible for depositing bone matrix, and to slow down the inflammatory processes responsible for the degeneration of the intervertebral disc.
Gli effetti biologici dell’ATP sono mediati dai recettori purinergici e, fra loro, P2X7R presenta caratteristiche molecolari e funzionali uniche. P2X7R è coinvolto in una serie di attività fisiologiche dell’apparato scheletrico, comprese solo in parte, oltre ad essere stato associato ad alcune malattie delle ossa e delle articolazioni, e, per queste ragioni, proposto come potenziale bersaglio terapeutico. Il lavoro di questa tesi si inserisce in un progetto più ampio finalizzato ad identificare nuovi target molecolari per lo sviluppo di strategie terapeutiche innovative nel trattamento di patologie che colpiscono ossa e cartilagini, e nel rallentamento del processo di degenerazione fisiologica dovuto all'invecchiamento. In linea con questo obiettivo, l’attenzione è stata anche rivolta alla messa a punto di modelli sperimentali in vitro aderenti al microambiente scheletrico in vivo, al fine di ottenere risultati particolarmente informativi. Lo studio si è rivolto prevalentemente a studiare: - la partecipazione di P2X7R nella maturazione delle cellule deputate alla deposizione di matrice mineralizzata cellulare. Cellule staminali da gelatina di Wharton del cordone ombelicale (hWJ-MSCs) e osteoblasti ottenuti da frammenti di tessuto osseo (hOBs) sono stati coltivati in condizioni di ipossia e normossia, trattati con antagonisti di P2X7R e apirasi (scavenger di ATP e ADP). I risultati ottenuti ci hanno permesso di attribuire un ruolo a P2X7R non tanto nel processo differenziativo a carico delle cellule staminali quanto nella capacità degli hOBs di depositare matrice mineralizzata. Inoltre, nell’indagare il possibile ruolo di fattori di trascrizione osteogenici nella modulazione dell'espressione di P2X7R, i dati di immunoprecipitazione della cromatina e trasfezione con vettori di espressione specifici hanno dimostrato: a. la presenza nel promotore del gene di P2RX7 di diversi siti di legame per fattori di trascrizione osteogenici; b. il reclutamento del fattore NFATc1 in siti specifici del promotore di P2X7R; c. l’aumento dell’espressione di P2X7R in seguito all’over espressione di NFATc1. - la partecipazione di P2X7R nell’omeostasi del disco intervertebrale (IVD). I meccanismi che sostengono l’insorgenza e la progressione dell’alterazione del microambiente dell’IVD sono ancora poco compresi. E’noto che l’ATP extracellulare è in grado di attivare l’inflammosoma NLRP3 con la partecipazione di P2X7R e il rilascio di IL-lβ. Questa citochina si ritiene sia l’evento chiave che contribuisce alla degenerazione del disco. Nello studio di questa tesi 83 campioni di IVD con diverso grado di degenerazione (Pfirrmann I-V) sono stati raccolti dopo l’intervento chirurgico, e impiegati sia per l’analisi istologica che per la realizzazione di colture primarie di cellule discali. I dati finora ottenuti hanno dimostrato che: a. il livello di degenerazione tissutale o sdifferenziamento cellulare correla con l’aumentata espressione di P2X7R e NLRP3; b. il rilascio di IL-1β è condizionata dalla presenza di LPS e BzATP (agonista del P2X7R), indicando un coinvolgimento dell’asse P2X7R-NLRP3. In conclusione è dimostrato che il P2X7R ed il suo target (NLRP3) sono coinvolti nella patogenesi della degenerazione del disco intervertebrale. Nell’insieme, i dati ottenuti suggeriscono che P2X7R rappresenti un buon bersaglio molecolare sia per promuovere l’attività anabolica delle cellule dell’osso deputate a depositare matrice ossea, sia per rallentare i processi infiammatori responsabili della degenerazione del disco intervertebrale.
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24

Bianco, F. "The role of P2X7 receptor in glial cells." Doctoral thesis, Università degli Studi di Milano, 2007. http://hdl.handle.net/2434/63133.

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Summary of thesis The aim of this study was to study the functional consequences of the ATP mediated communication between astrocytes and microglia, with particular emphasis on the functional consequences related to the activation of purinergic receptor P2X7 on microglial cells. The results obtained indicate that: 1. that the LPS-induced reduction of functional P2X7 receptor in embryonic microglia may mediate the cell cycle modifications observed upon exposure to the endotoxin. 2. the release of at least a fraction of IL 1-beta from microglia takes place, upon ATP stimulation, through the shedding of vesicles from the plasmamembrane. Shed vesicles appear to be the site of IL 1-beta processing. 3. that intercellular calcium mediated signalling might contribute in cortex more than in the hippocampus to the propagation and maintenance of an inflammatory state. Most importantly, the present study represents the first evidence of microvesicle shedding in the CNS. Just like their systemic counterpart (monocytes) microglial cells are able to form vesicle and release them in a calcium-dependent manner. We have demonstrated that vesicle contain protein elements and that biological processes such as IL 1-beta maturation can occur inside isolated vesicles.
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25

Huang, Szu-Wei. "The role of the purinergic P2X7 receptor in small intestinal inflammation." Thesis, University of Manchester, 2015. https://www.research.manchester.ac.uk/portal/en/theses/the-role-of-the-purinergic-p2x7-receptor-in-small-intestinal-inflammation(e96a14cf-de69-47ac-bcf9-7730d9006364).html.

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The purinergic P2X7 receptor (P2X7R), an adenosine triphosphate (ATP)-gated receptor, is widely distributed in a variety of cell types such as neuron cells, immune cells and epithelial cells. P2X7R on cells senses extracellular ATP released from dying cells which then acts as a danger signal and initiates inflammation. Activation of P2X7R results in various downstream events, including Ca2+ influx, nonselective membrane pore formation, cell death, assembly of the inflammasome, and killing of intracellular pathogens. Epithelial cells in the gut also express P2X7R and act as a sentinel that protects against infection and responds to changes in environmental stimuli. However, the role of P2X7R in IECs is poorly defined. Given that infection of pathogens often causes cellular damage and the released ATP may be sensed by P2X7R, we hypothesised that IECs initiate intestinal inflammation via activation of the P2X7R in response to infection. Thus, the aim of this thesis was to characterise the role of P2X7R in the initiation and development of small intestinal inflammation. In order to achieve this aim, we used two parasite-induced murine ileitis models, Toxoplasma gondii (T. gondii) and Trichinella spiralis (T. spiralis), which induce Th1 and Th2 immunity respectively. In the in vivo model of T. gondii infection, we found that P2X7R deficiency was associated with less intestinal epithelial responsiveness to the infection. The P2X7R-/- IECs had reduced CCL5 and CCL20 chemokine expression which was associated with reduced recruitment of CD103+CD11b- dendritic cells (DCs) to the small intestinal epithelium at day 1 post infection (p.i.). This finding was supported by infection of bone marrow chimeras showing a decrease in the recruitment of WT P2X7R+/+ CD103+ DCs to a P2X7R-/- epithelium. To address whether the reduced DC response impacted on development of adaptive immunity, we analysed serum IFN-g and the proportion of splenic IFN-g+CD4+ T cells at day 8 p.i., and showed they were reduced in P2X7R-/- mice. In the in vivo model of T. spiralis infection, P2X7R deficiency was also associated with reduced intestinal epithelial responsiveness to this infection characterised by lower CCL5 expression in IECs. A significant decrease in the recruitment of CD103+CD11b+ DCs at day 2 p.i. was noted in P2X7R-/- animals, and the importance of epithelial P2X7R in DC recruitment was confirmed using bone marrow chimeras. The P2X7R-/- mice, compared with the WT, had delayed progression of small intestinal inflammation, accompanied by a reduction in the percentage of IL-4+CD4+ T cells and IL-4 levels at day 8 p.i. The reduced IL-4 response was associated with a delayed worm expulsion in the P2X7R-/- mice at day 12 p.i. An in vitro study demonstrated that P2X7R blockade using the chemical inhibitor A-740003, significantly decreased CCL5, IL-6 and TNF-a secretion from mouse intestinal epithelial CMT-93 cells in response to T. gondii infection. A similar decrease in the level of CCL5 produced was also observed using primary P2X7R-/- intestinal crypt cells stimulated with lipopolysaccharide (LPS) compared with WT cells. This data indicates a proinflammatory role for P2X7R during infection. Although P2X7R signalling is known to induce the assembly of the inflammasome, IECs did not secrete the inflammasome-associated cytokines IL-1b and IL-18 in response to T. gondii infection. Moreover, P2X7R signalling had no effect on the induction of cell death in T. gondii-infected IECs. Interestingly, there was a novel finding that P2X7R antagonism inhibited T. gondii infectivity in CMT-93 cells. In summary, we have shown that P2X7R signalling mediated CCL5 expression in IECs in response to infection. Epithelial chemokines are important for the initiation of small intestinal inflammation via recruitment of innate cells such as DCs which can then prime for protective adaptive immunity. These results in this thesis improve the understanding of the role of P2X7R in the intestinal immune system and reveal novel roles for epithelial P2X7R. The work suggests the potential of epithelial P2X7R as a target for pharmacological treatment of intestinal inflammatory disorders.
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26

Robinson, Lucy Elizabeth. "Regulation of the P2X7 receptor by plasma membrane cholesterol." Thesis, University of Cambridge, 2015. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.709439.

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27

Chavda, S. "P2X7 receptor modulation of visual responses in the retina." Thesis, University College London (University of London), 2015. http://discovery.ucl.ac.uk/1464069/.

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Adenosine 5’ triphosphate (ATP)-gated P2X7 receptors (P2X7Rs) are known to act as conduits for photoreceptor and retinal ganglion cell (RGC) damage, consequences of various neurodegenerative conditions within the visual pathway. Growing evidence supports the notion that P2X7Rs and associated inflammatory mediators may coordinate microglia, the resident immune cells of the central nervous system (CNS), to play a genuine role in modulating neurotransmission. This study aimed to characterise the role of P2X7Rs in modulating outer and inner retinal function within the rod-mediated pathway, and in the putative microglial-mediated modulation of signal transmission in the retina. Excitatory components of outer and inner retinal function were assessed by recording light-evoked, extracellular transretinal electroretinogram (ERG), and ON and OFF retinal ganglion cell (RGC) field excitatory postsynaptic potential (fEPSP) responses from the acutely isolated, dark-adapted, mouse retina. Alterations to microglial morphology, under similar conditions, were also explored. Initial experiments confirmed the excitatory responses as predominantly mediated via the ‘classic’ rod photoreceptor – rod-ON bipolar cell – AII amacrine cell pathway. With the use of selective P2X7R antagonists, it was shown that P2X7R activation directly modulated photoreceptor, ON bipolar cell and ON RGC function, but not OFF RGC function, through partially independent mechanisms. A novel finding of this study demonstrated that acute application of the microglia-activating bacterial component, lipopolysaccharide (LPS) modulated inner retinal function, possibly through a P2X7R- and Pannexin-1-associated mechanism of microglial ATP release. These results were supported by observations of early morphometric changes to microglia caused by P2X7R activation and LPS, as revealed by immunofluorescence labelling and confocal laser scanning microscopy. Since changes in neurotransmission and microglial function are early indicators of neuropathology, these results contribute to the understanding of early neural-immune interactions in retinal disease, and in the central nervous system as a whole.
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Gonnord, Pauline. "Etude des relations structure-fonction du récepteur purinergique P2X7." Paris 11, 2008. http://www.theses.fr/2008PA11T024.

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Callegari, Maria Giulia <1978&gt. "Ruolo del recettore P2X7 nella sopravvivenza e morte cellulare." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2007. http://amsdottorato.unibo.it/259/1/TESI_Maria_Giulia_Callegari.pdf.

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30

Callegari, Maria Giulia <1978&gt. "Ruolo del recettore P2X7 nella sopravvivenza e morte cellulare." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2007. http://amsdottorato.unibo.it/259/.

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31

Besnard, Aurore. "Rôle de la signalisation purinergique au cours de la régénération du foie chez la souris : étude des récepteurs P2X4 et P2X7." Paris 6, 2013. http://www.theses.fr/2013PA066054.

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Objectif : La régénération hépatique est un processus complexe qui met en jeu de nombreux signaux régulateurs endocrines, paracrines, autocrines et nerveux. L’ATP extracellulaire et plus généralement la signalisation purinergique, ont été décrits comme ayant un rôle dans la survie et la prolifération cellulaires ainsi que dans les processus inflammatoires et sécrétoires. Le laboratoire a rapporté antérieurement que l’ATP extracellulaire contribuait à la régénération du foie chez le rat. L’objectif de ce travail était d’étudier le rôle des récepteurs ionotropiques P2X4 et P2X7 au cours de la régénération hépatique après hépatectomie des deux tiers (Hx) chez la souris. Résultats : Les récepteurs P2X4 et P2X7 étaient fortement exprimés par les cellules de Kupffer et par les hépatocytes avec, pour le récepteur P2X4, un renforcement canaliculaire et sous-canaliculaire. Après Hx, un retard de régénération par rapport aux souris WT (restauration de la masse du foie, expression protéique de la cycline D1 et du PCNA, entrée en mitose des hépatocytes), ainsi que des lésions de nécrose hépatique et une cholestase prolongée étaient observés chez les souris P2X4 KO mais pas chez les P2X7 KO. La réponse adaptative hépatocytaire à la surcharge en acides biliaires après Hx n’était pas altérée chez les souris P2X4 KO (vs WT) (régulation transcritpionnelle de CYP7a1, NTCP, et OSTb), alors que l’adaptation du flux biliaire et de la sécrétion d’HCO3- dans la bile était anormale. Enfin, une réponse inflammatoire exacerbée était observée après Hx chez les souris P2X4 KO, (ARNm et concentrations plasmatiques de IL-1β, TNF-α et IL-6) par rapport aux souris WT. In vitro, le récepteur P2X4 n’avait pas d’impact significatif sur la prolifération hépatocytaire, ni sur la réponse au LPS (lipopolysaccharide) ou à l’ATP des macrophages péritonéaux (MP). Conclusion : Pendant la régénération du foie, le récepteur P2X4 contribuerait au contrôle de l’homéostasie bilaire et de la réponse inflammatoire, deux éléments dont la régulation est essentielle au bon déroulement du processus de régénération. Les mécanismes par lesquels le récepteur P2X4 régule ces différents processus restent à déterminer
Background : Liver regeneration is a complex process during which various endocrine, paracrine, autocrine and nervous factors play important roles. Extracellular ATP and more generally purinergic signalling has been described to regulate cell survival and proliferation, as well as inflammatory processes. We previously reported that extracellular ATP contributed to liver regeneration in the rat. In this work, we analysed the involvement of the membrane ionotropic P2X4 and P2X7 purinergic receptors during liver regeneration after a two-third partial hepatectomy (PH) in mice. Results : P2X4 and P2X7 receptors were highly expressed in Kupffer cells, and in hepatocytes with reinforcement in the sub-canalicular and canalicular areas for P2X4 receptor. After PH, there was a delay in P2X4 KO as compared to WT mice, in liver mass restoration, cyclin D1 and PCNA expression, and mitotic activity. Post-PH hepatocyte necrosis (periportal focal “bile infarcts”) and prolonged cholestasis were observed in P2X4-KO mice, but neither WT, nor P2X7 KO mice. Adaptive response to post-PH cholestasis (CYP7a1, NTCP and OSTb mRNA regulation) was similar in WT and P2X4-KO livers. In P2X4 KO mice after PH, as compared with WT, smaller increase in bile flow and HCO3- biliary output were observed. Early mRNA induction, as well as plasma concentration rise in cytokines (IL1 β, TNFα and IL6) were greater in P2X4-KO than WT mice after PH. In vitro, the P2X4 receptor didn’t impact significantly hepatocyte proliferation, nor peritoneal macrophages (PM) inflammatory reponse to LPS (lipopolysaccharide) or ATP. Conclusions : During liver regeneration, P2X4 may contribute to the complex control of hepatocyte proliferation through the regulation of biliary homeostasis and inflammation. Mechanisms underlying P2X4 involvement in those processes still remain to be defined
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32

Teodoro, Luis Henrique de Souza. "Caracterização funcional do receptor P2X7 na glândula pineal de rato." Universidade de São Paulo, 2013. http://www.teses.usp.br/teses/disponiveis/41/41135/tde-28032014-093312/.

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A sinalização purinérgica tem sido demonstrada como um um importante modulador dediversos processos fisiológicos e fisiopatológicos. Dentre os receptores purinérgicos, o receptor P2X7 distingui-se por requerir altas concentrações de ATP em sua ativação. As demonstrações prévias de que a glândula pineal responde a diferentes estímulos purinérgicose a altas concentrações de ATP sugere um papel para os receptores P2X7 nesta glândula, embora sua expressão e função não estivesse estabelecida. O objetivo deste trabalho, portanto, foi caracterizar funcionalmente o receptor P2X7 na glândula pineal de ratos. Os resultados obtidos demonstram, pela primeira vez, a expressão gênica e proteica do receptor P2X7 na glândula pineal. Os efeitos da ativação destes receptores levam a uma inibição nos níveis de melatonina induzida por isoprenalina por um mecanismo independente da via do fator de transcrição NF-kB e da fosfolipase C. Além disso, a estimulação destes receptores inibiu a síntese de TNF induzida por LPS, resultado este semelhante ao observado na presença do pré-tratamento com antagonistas do receptor P2X7. Estes dados confirmam a presença de receptores P2X7 na glândula pineal e reiteram o relevante papel da estimulação purinérgica a sobre a síntese de melatonina e sobre a capacidade da pineal em responder a PAMPs, como o LPS
Purinergic signalling has been demonstrated as an important modulator ofseveral physiological and pathophysiological processes. Among the purinergic receptors, the activation of P2X7 receptor requireshigh concentrations of ATP. The previous demonstration that the pineal gland is responsive to different purinergic stimulus and to high concentrations of ATPsuggests a role for P2X7, although its expression and function remained unclear. The aim of this study was to functionally characterize the P2X7 receptor in the rat pineal gland.The data showedthe P2X7 receptor mRNA and protein expression in the pineal gland. The effect of its activation leads toan inhibition of melatonin content induced by isoprenaline through an independent NF-kB and PLC pathways. Furthermore, the P2X7 receptor activation inhibits the LPS-induced TNF synthesis, a similar result observed in the presence of the pre-treatment with P2X7 receptors antagonists. These data demonstrate the presence of P2X7 receptors in the rat pineal gland and confirm the relevant role of the purinergic stimulation to the pineal melatonin synthesis and responsiveness to PAMPs such as LPS
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33

Teixeira, Juliana Maia 1984. "Involvement of P2X3 and P2X7 purinergic receptors in inflammatory articular hyperalgesia in the knee joint of rats and the study of the peripheral mechanisms involved = Participação dos receptores purinérgicos P2X3 e P2X7 na hiperalgesia inflamatória articular em joelho de ratos e o estudo dos mecanismos periféricos envolvidos." [s.n.], 2014. http://repositorio.unicamp.br/jspui/handle/REPOSIP/314054.

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Orientador: Cláudia Herrera Tambeli
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: A osteoartrite (OA) é uma doença degenerativa e progressiva, caracterizada pela degradação da cartilagem que reveste as extremidades ósseas e inflamação da membrana sinovial, causando incapacidade física, inchaço articular e dor. Embora o alívio da dor severa seja o principal objetivo no tratamento agudo, pouco se sabe sobre os mecanismos envolvidos no desenvolvimento da dor na OA. Estudos demonstram a participação do ATP (adenosina 5¿-trifosfato) em processos de hiperalgesia através da ativação dos receptores purinérgicos P2X3, P2X2/3 e P2X7. Portanto, os objetivos deste estudo foram: (1) investigar a participação dos receptores P2X3, P2X2/3 e P2X7 na hiperalgesia articular em modelo de artrite na articulação do joelho de ratos machos e fêmeas em estro e se há diferenças sexuais no efeito induzido pelos antagonistas de receptores P2X3, P2X2/3 e P2X7. (2) testar a hipótese de que a inflamação articular induzida pela carragenina aumenta a expressão do receptor P2X3 nos condrócitos da cartilagem articular da articulação do joelho de ratos. (3) verificar se o mecanismo pelo qual a ativação dos receptores P2X3, P2X2/3 e P2X7 contribui para a hiperalgesia articular depende da liberação prévia de citocinas pró-inflamatórias e da migração de neutrófilos. (4) investigar se a ativação dos receptores P2X3, P2X2/3 e P2X7 induz hiperalgesia na articulação do joelho de ratos dependente da liberação de mediadores inflamatórios. (5) testar a hipótese de que a ativação dos receptores P2X3, P2X2/3 e P2X7 contribui para a hiperalgesia articular induzida pelos mediadores inflamatórios: bradicinina, citocinas pró-inflamatórias, PGE2 e dopamina. Para os objetivos 1, 4 e 5, a hiperalgesia articular foi quantificada através do teste de Incapacitação Articular. Para o objetivo 2, foi utilizado o ensaio de imunofluorescência. Para os objetivos 3 e 4 foram utilizados os ensaios imuno-enzimáticos ELISA e MPO. Os resultados demonstram que a ativação dos receptores P2X3, P2X2/3 e P2X7 pelo ATP endógeno é essencial para o desenvolvimento da hiperalgesia articular induzida pela carragenina na articulação do joelho de ratos machos e fêmeas em estro, que são mais sensíveis do que os machos aos efeitos anti-hiperalgésicos e anti-inflamatórios induzidos pelo antagonista de receptor P2X7. Durante a inflamação articular induzida pela carragenina ocorre um aumento na expressão dos receptores P2X3 nos condrócitos da cartilagem articular. O papel dos receptores P2X3, P2X2/3 e P2X7 na hiperalgesia articular é mediado pela sensibilização indireta dos nociceptores aferentes primários, dependente da liberação prévia de citocinas pró-inflamatórias e da migração de neutrófilos. Além disso, a ativação dos receptores P2X3, P2X2/3 e P2X7 induz hiperalgesia articular dependente da liberação de bradicinina, aminas simpatomiméticas, prostaglandinas e citocinas pró-inflamatórias. Finalmente, a hiperalgesia articular induzida pelos mediadores inflamatórios bradicinina, PGE2 e dopamina depende da ativação de receptores P2X3 e P2X2/3, enquanto que a ativação de receptor P2X7 contribui para a hiperalgesia articular induzida pela bradicinina e dopamina. Concluindo, os resultados apresentados sugerem que os receptores P2X3, P2X2/3 e P2X7 são alvos farmacológicos interessantes para o tratamento das doenças inflamatórias articulares como a osteoartrite. Particularmente em relação ao receptor P2X7, antagonistas seletivos podem ser usados para reduzir a dor e inflamação no joelho, especialmente em mulheres
Abstract: Osteoarthritis (OA) is a degenerative and progressive disease, characterized by cartilage breakdown which covers the bone ends and by synovial membrane inflammation, causing disability, joint swelling and pain. The relief of severe pain is the main goal of the acute treatment, but little is known about the mechanisms involved in the development of pain in OA. It has been demonstrated the role of ATP (adenosine 5'-triphosphate) in processes of hyperalgesia through activation of purinergic receptors P2X3, P2X2/3 and P2X7. Therefore, the aims of this study were: (1) to investigate the role of P2X3, P2X2/3 and P2X7 receptors in articular hyperalgesia in the knee joint arthritis model in males and estrus females rats and, if so, whether there are sex differences in the effect induced by the selective P2X3, P2X2/3 and P2X7 receptors antagonists. (2) to test the hypothesis that the carrageenan-induced articular inflammation increases the expression of P2X3 receptor in chondrocytes of articular cartilage of the knee joint. (3) to verify whether the mechanism by which the P2X3, P2X2/3 and P2X7 receptors activation contributes to articular hyperalgesia depends on previous pro-inflammatory cytokines release and neutrophil migration. (4) to investigate whether the P2X3, P2X2/3 and P2X7 receptors activation induces articular hyperalgesia in the rat¿s knee joint which depends on release of inflammatory mediators. (5) to verify whether the activation of P2X3, P2X2/3 and P2X7 receptors contributes to the articular hyperalgesia induced by the inflammatory mediators bradykinin, pro-inflammatory cytokines, PGE2 and dopamine. For the aims 1, 4 and 5, the articular hyperalgesia was quantified by the rat knee joint Incapacitation Test. The immuno?uorescence method was used for the aim 2. For aims 3 and 4, the ELISA and MPO immunoenzymatic assays were used. The results demonstrate that P2X3, P2X2/3 and P2X7 receptors activation by endogenous ATP is essential for the development of carrageenan-induced articular hyperalgesia in the knee joint of male and estrus female rats, which are more sensitive than males to anti-hyperalgesic and anti-inflammatory effects induced by the P2X7 receptor antagonist. During carrageenan-induced joint inflammation occurs an increased of P2X3 receptors expression in chondrocytes of the articular cartilage. The essential role played by P2X3, P2X2/3 and P2X7 receptors in the development of articular hyperalgesia is mediated by an indirect sensitization of the primary afferent nociceptors dependent on the previous pro-inflammatory cytokines release and neutrophil migration. Moreover, the P2X3, P2X2/3 and P2X7 receptors activation induces articular hyperalgesia which depends on bradykinin, sympathomimetic amines, prostaglandins and pro-inflammatory cytokines release. Finally, the articular hyperalgesia induced by inflammatory mediators bradykinin, PGE2 and dopamine depends on the P2X3 and P2X2/3 receptors activation, while the P2X7 receptor activation contributes to the bradykinin- and dopamine-induced articular hyperalgesia. In conclusion, our results suggest that P2X3, P2X2/3 and P2X7 receptors are interesting pharmacological targets for the treatment of inflammatory joint diseases such as osteoarthritis. In particular, selective P2X7 receptor antagonists can be used to reduce inflammation and pain in the knee joint, especially in women
Doutorado
Fisiologia
Doutora em Biologia Funcional e Molecular
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34

Xu, Xing Jian. "Characterization of the functional properties of P2X7 receptor splice variants." Thesis, University of Cambridge, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.610293.

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35

Fischer, Wolfgang, Heike Franke, Ute Krügel, Heiko Müller, Klaus Dinkel, Brian Lord, Michael A. Letavic, David C. Henshall, and Tobias Engel. "Critical evaluation of P2X7 receptor antagonists in selected seizure models." Universitätsbibliothek Leipzig, 2016. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-206115.

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The ATP-gated P2X7 receptor (P2X7R) is a non-selective cation channel which senses high extracellular ATP concentrations and has been suggested as a target for the treatment of neuroinflammation and neurodegenerative diseases. The use of P2X7R antagonists may therefore be a viable approach for treating CNS pathologies, including epileptic disorders. Recent studies showed anticonvulsant potential of P2X7R antagonists in certain animal models. To extend this work, we tested three CNS-permeable P2X7R blocker (Brilliant Blue G, AFC-5128, JNJ-47965567) and a natural compound derivative (tanshinone IIA sulfonate) in four well-characterized animal seizure models. In the maximal electroshock seizure threshold test and the pentylenetetrazol (PTZ) seizure threshold test in mice, none of the four compounds demonstrated anticonvulsant effects when given alone. Notably, in combination with carbamazepine, both AFC-5128 and JNJ-47965567 increased the threshold in the maximal electroshock seizure test. In the PTZ-kindling model in rats, useful for testing antiepileptogenic activities, Brilliant Blue G and tanshinone exhibited a moderate retarding effect, whereas the potent P2X7R blocker AFC-5128 and JNJ-47965567 showed a significant and long-lasting delay in kindling development. In fully kindled rats, the investigated compounds revealed modest effects to reduce the mean seizure stage. Furthermore, AFC-5128- and JNJ-47965567-treated animals displayed strongly reduced Iba 1 and GFAP immunoreactivity in the hippocampal CA3 region. In summary, our results show that P2X7R antagonists possess no remarkable anticonvulsant effects in the used acute screening tests, but can attenuate chemically-induced kindling. Further studies would be of interest to support the concept that P2X7R signalling plays a crucial role in the pathogenesis of epileptic disorders.
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36

Gartland, Alison. "The role of the P2X7 receptor in bone cell formation." Thesis, University of Liverpool, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.343968.

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37

Caseley, Emily Alice. "Structural basis for ligand-receptor interactions at the P2X7 receptor." Thesis, University of Leeds, 2016. http://etheses.whiterose.ac.uk/15858/.

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P2X7 receptors (P2X7Rs) belong to the P2X receptor family of ligand-gated ion channels activated by extracellular ATP. The human P2X7R (hP2X7R) is implicated in numerous debilitating disease conditions and thus represents a promising therapeutic target. However, P2X7R structure-function relationships remain less well understood. The study presented in this thesis used electrophysiology in conjunction with structural modelling, molecular docking and site-directed mutagenesis to better understand the structural basis for ligand-receptor interactions at P2X7Rs and used such structural information to identify novel hP2X7R antagonists. Initially, P2X7R homology models were produced based on the crystal structures of the zebrafish P2X4R (zfP2X4R) in closed and ATP-bound states and validated through docking and biochemical approaches. First of all, molecular docking showed that ATP binds to the zfP2X4R and hP2X7R in a strikingly similar configuration to the crystal structure. Secondly, docking of the antagonists AZ11645373, KN62 and SB203580 revealed a specific interaction with Phe95 in the hP2X7R that is absent in the rat P2X7R (rP2X7R), providing structural insight into their preferential hP2X7R antagonism. Thirdly, replacing Asp48 and Ile331 with cysteine resulted in disulfide bonding that impaired hP2X7R-mediated currents, which was reversibly restored by dithiothreitol. These results are consistent with the transmembrane domains moving substantially apart, as predicted by the closed and open state models. Overall, these experiments show that homology models can yield meaningful structural information in terms of P2X7R interactions with ATP, antagonists and receptor activation. The second part of the study searched for P2X7R antagonists using a structure-based approach. Virtual screening of ~100,000 compounds in the ZINC library against the ATP-binding pocket in the hP2X7R model identified C23, C40 and C60 as structurally novel antagonists of the hP2X7R but not the rP2X7R. These compounds inhibited the agonist-evoked increase in intracellular Ca2+ concentration ([Ca2+]i) with IC50 values in the micromolar range. C23 and C40 also inhibited agonist-induced currents with similar potency, but C60 did not. All three compounds suppressed large pore formation with micromolar potency. While C23 inhibited agonist-induced [Ca2+]i increase mediated by the hP2X4R and rP2X3R, C40 and C60 were more selective towards the hP2X7R. In conclusion, these results show C23, C40 and C60 as novel hP2X7R antagonists. Such structure-based approaches should aid novel P2XR antagonist identification. Finally, the models were used in combination with site-directed mutagenesis to investigate residues influencing hP2X7R-agonist interactions. The first set of experiments examined four residues implicated in interactions with ATP, and only the mutation of Tyr288 to various residues significantly affected agonist sensitivity. This Tyr288 mutation abolished receptor function, which was mainly due to impairment in protein surface expression as shown by immunofluorescent imaging. The second set of experiments examined several residues for their contribution in the difference in functional expression and agonist sensitivity. Substitution of Val87 in the hP2X7R for Ile in the rP2X7R increased the maximal agonist-induced currents. Overall, the present study provides structural insights into ligand-receptor interactions at the P2X7Rs. It demonstrates that structure-based approaches are feasible in identifying novel antagonists, and this has wider implications for the P2XR family and membrane proteins as a whole.
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38

Jelassi, Bilel. "Implication des récepteurs P2X7 dans l'invasivité des cellules cancéreuses humaines." Thesis, Tours, 2013. http://www.theses.fr/2013TOUR3318/document.

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Le récepteur-canal P2X7 est fortement exprimé et est fonctionnel dans la lignée de cellules cancéreuses mammaires humaines hautement invasives MDA-MB-435s. L’activation de P2X7 par l’ATP extracellulaire est responsable de l'émission des prolongements cellulaires et l'augmentation de la migration cellulaire. En outre, l’activation de P2X7 augmente l’invasion cellulaire à travers la matrice extracellulaire et fait intervenir la libération de forme mature de cathepsines à cystéine dans le milieu extracellulaire. L’inhibition pharmacologique de P2X7 diminue l’invasivité des cellules cancéreuses dans un modèle de micrométastases chez le poisson zèbre. Nous avons également montré que l’émodine (1,3,8-trihydroxy-6-méthylanthraquinone) une anthraquinone isolée de Rheum officinale Baill (Rhubarbe chinoise) inhibe l’invasivité des cellules cancéreuses humaines via l’antagonisme de P2X7 et n’as pas d’effet sur les autres récepteurs P2X. Nos résultats démontrent un nouveau mécanisme entre la fonctionnalité de P2X7 dans les cellules cancéreuses et l’invasivité cellulaire, un paramètre clé dans la croissance tumorale et le développement des métastases. Ceci suggère également un rôle thérapeutique potentiel pour les antagonistes des P2X7
P2X7 receptor channel is highly expressed and fully functional in the highly invasive human breast cancer cell line MDA-MB-435s. Its activation by extracellular ATP is responsible for the extension of neurite-like cellular prolongations, and the increase in cell migration. Furthermore, P2X7 activation enhanced invasion through the extracellular matrix and was related to the increase of mature forms of cysteine cathepsins in the extracellular medium. Pharmacological targeting of P2X7 decreases cancer cell invasiveness in a zebrafish model of micrometastases. We also showed that emodin (1,3,8-trihydroxy-6-methylanthraquinone) an anthraquinone derivative originally isolated from Rheum officinale Baill (Chinese Rhubarb) inhibits human cancer cell invasiveness by specifically antagonizing the P2X7 and not the other members of the P2X family. Our results demonstrate a novel mechanistic link between P2X7 functionality in cancer cells and invasiveness, a key parameter in tumour growth and in the development of metastases. These results also suggest a potential therapeutic role for the newly developed P2X7 antagonists
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39

Soares, Rafael Ferreira. "Estudos in silico do comportamento dinâmico do receptor P2x7 humano." reponame:Repositório Institucional da FIOCRUZ, 2015. https://www.arca.fiocruz.br/handle/icict/13939.

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Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil
O receptor P2X7, contido na família de receptores P2, é um receptor inotrópico purinérgico, cátion seletivo, ativado por nucleotídeos (ATP). Esses receptores são expressos em células do sistema nervoso central, periférico e células da linhagem hematopoiética além de estarem envolvidos em diversos processos biológicos como a transdução de sinais da dor, participação na inflamação granulomatosa, dentre outros. Dos seus sete subtipos (P2X1 \2013 P2X7) o P2X7 possui a característica de formar um poro cuja condutância é aumentada em aproximadamente 26 vezes em relação à condutância padrão de todos os receptores da família P2X (~15 pS). Este aumento na condutância se reflete em possibilitar a passagem de moléculas de até 900 Da. Este estudo propõe através da aplicação das técnicas de modelagem comparativa e dinâmica molecular analisar o comportamento dinâmico de um modelo do receptor P2X7 humano, desde seus movimentos de fechamento e abertura do canal de baixa condutância, até a passagem de íons catiônicos (Potássio) e aniônicos (cloreto) para avaliar a seletividade do canal. Isto é necessário pois ainda não se dispõe de um modelo experimental (Ressonância Magnética Nuclear ou Cristalografia de raio-X) e ainda existem dúvidas sobre o mecanismo de ativação que elucide o processo de transição entre o estado de menor condutância (um canal aberto) para o estado de maior condutância (formação de um poro) Foram construídos dois modelos do receptor P2X7 por meio de modelagem comparativa utilizando como molde o receptor P2X4 do organismo Danio rerio (único molde viável até o momento) em seus estados aberto e fechado. As simulações de dinâmica molecular revelaram o tempo de fechamento de canal (região transmembranar) e o rearranjo do sítio de ligação ao ATP em ~400ps e ~650ps, respectivamente. As dinâmicas de passagem dos íons confirmaram as características cátion seletivas do canal, visto que foi possível observar a passagem de todos os íons de potássio dispostos na região de estreitamento do canal e também foi observada a expulsão de uma das três dispostas na mesma região que os íons catiônicos avaliados. Estes resultados ressaltam que os sistemas simulados para avaliar o comportamento do receptor P2X7 descrevem comportamentos semelhantes aos descritos experimentalmente ressaltando a possibilidade de uso dos modelos construídos neste trabalho para aplicação em estudos piloto futuros (virtual screening, docking molecular) para avaliação de compostos promissores que atuem como agonistas ou antagonistas mais seletivos que os compostos existentes atualmente
The P2X7 is an inotropic, purinergic, cation-selective and activated by nucleotides (ATP) receptor that belongs to the P2 receptor family. These receptors are found in central and peripheral nervous system cells and in hematopoietic lineage cells. In addition, P2X7 receptors are involved in a vast number of biological processes such as pain signal transduction, participation in granulomatous inflammation, among others. From all seven subtypes (P2X1 \2013 P2X7), P2X7 receptor presents the characteristic of forming a pore whose conductance increases approximately 26 times more than the standard conductance from all others P2X receptors (~15 pS). This rise in the conductance allows the entrance of molecules up to 900 Da. The aim of this study is to employ comparative modeling and molecular dynamics methods to analyze the dynamic behavior of the human P2X7 receptor to assess the channel selectivity, regarding the opening and closing movements and the passage of cations (potassium) and anions (chloride) across the low conductance channel). The combination of these methods is necessary since there is no model determined by experimental methods such as Nuclear Magnetic Resonance or X-ray Crystallography, and doubts remain about the activation mechanism that drives the receptor form a state of low conductance (open channel) to a state of high conductance (pore formation) Two P2X7 receptor models were built by means of comparative modeling using as template the 3D structure of P2X4 receptor from Danio rerio organism in its open and close states. Molecular dynamics simulations revealed the channel closing time (transmembrane region) and the rearrangement of the ATP binding site took place, in ~400 ps and ~650 ps, respectively. The ion entry simulations confirmed the cation selectivity characteristics of the channel, since it was possible to observe the entrance of all potassium molecules present in the narrowed region of the channel. Furthermore, it was noted the expelling of the chloride molecules at the same regions. These results highlight that the P2X7model reproduce its experimental behavior. It is possible to describe similar mechanisms described experimentally; emphasizing the prospect of using these models in future studies (virtual screening, molecular docking) to evaluate lead compounds, capable of acting as more selective agonists or antagonist than the current available ones
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40

CAPECE, Marina. "Accelerated Tumor Progression in Mice Lacking the ATP Receptor P2X7." Doctoral thesis, Università degli studi di Ferrara, 2016. http://hdl.handle.net/11392/2403471.

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Il recettore per l’ATP, P2X7 (P2X7R) riveste un ruolo fondamentale nell’infiammazione e nell’immunità, ma la sua funzione in ambito tumorale non è ancora stata completamente stabilita. In questo studio abbiamo analizzato la crescita e la disseminazione tumorale di cellule murine di melanoma (B16) o colon carcinoma (CT26) in modelli in vivo deleti del gene per il recettore P2X7 (P2X7R-KO). La crescita della massa tumorale sottocute è stata valutata tramite la misurazione manuale con il calibro, mentre la trasfezione delle cellule con una luciferasi intracitosolica ci ha permesso di monitorare la disseminazione metastatica tramite il rilevamento in vivo dell’emissione di fotoni. Da queste osservazioni e da valutazioni post-mortem è risultato che, nei topi P2X7R-KO la crescita e la disseminazione tumorali hanno un decorso più accelerato rispetto a quelli di un topo wild-type (wt) per il P2X7R. Analisi ex-vivo sui tumori sperimentali, hanno mostrato che i livelli di IL-1β e VEGF, due importanti citochine, il cui rilascio è P2X7R-dipendente, sono più bassi nel topo P2X7R-KO, dove è quasi completamente assente anche l’infiltrato infiammatorio. La riduzione di cellule infiammatorie nel sito tumorale è dovuta all’assenza del recettore nell’ospite, come dimostrano esperimenti in cui cellule che non esprimono il recettore si dimostrano incapaci di rispondere alla stimolazione fatta con cellule tumorali o presentano una capacità migratoria ridotta rispetto alle cellule wt per P2X7R. L’importanza della funzionalità del recettore P2X7 delle cellule di derivazione ematopoietica nella progressione tumorale è stata confermata da esperimenti condotti su chimere di topi wt e P2X7R-KO. Topi wt trapiantati con cellule derivanti dal midollo osseo di topi P2X7R-KO hanno manifestato una crescita tumorale paragonabile a quella avvenuta nei topi P2X7R-KO. Complessivamente i nostri risultati dimostrano che l’espressione e la funzionalità del recettore P2X7 sono determinanti per supportare una efficace risposta immunitaria anti-tumorale.
The ATP receptor P2X7 (P2X7R) has a key role in inflammation and immunity, but its possible roles in cancer are not firmly established. In the present study, we investigated the effect of host genetic deletion of P2X7R in the mouse on the growth of B16 melanoma or CT26 colon carcinoma cells. Tumor size and metastatic dissemination were assessed by in vivo caliper and luciferase luminescence emission measurements along with post-mortem examination. In P2X7R-deficient mice, tumor growth and metastatic spreading were accelerated strongly, compared with wild-type (wt) mice. Intra-tumoral IL-1β and VEGF release were drastically reduced, and inflammatory cell infiltration was abrogated nearly completely. Similarly, tumor growth was also greatly accelerated in wt chimeric mice implanted with P2X7R-deficient bone marrow cells, defining hematopoietic cells as a sufficient site of P2X7R action. Finally, dendritic cells from P2X7R-deficient mice were unresponsive to stimulation with tumor cells, and chemotaxis of P2X7R-less cells was impaired. Overall, our results showed that host P2X7R expression was critical to support an antitumor immune response, and to restrict tumor growth and metastatic diffusion
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41

Mendes, Cristina Eusébio. "Estudo das células gliais entéricas imunorreativas aos receptores P2x2 e P2x7 do íleo de ratos submetidos à isquemia e reperfusão intestinal." Universidade de São Paulo, 2013. http://www.teses.usp.br/teses/disponiveis/42/42131/tde-13032014-173541/.

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A resposta do sistema nervoso para diversas lesões acarreta a ativação das células gliais entéricas. Este trabalho tem como objetivo analisar o efeito da isquemia e reperfusão intestinal (I/R-i) sobre as células gliais entéricas, neurônios e receptores P2X2 e P2X7. Foram analisados o íleo de ratos Controle, Sham e I/R-i com 0 hora, 24 horas e 14 dias de reperfusão. Foram realizadas dupla marcação dos receptores P2X2 e P2X7 com Hu e S100, densidade, área do perfil e marcação de proliferação celular. Os resultados mostraram dupla marcação de células gliais entéricas e neurônios com os receptores P2X2 e P2X7; a densidade apresentou um aumento de células gliais e diminuição de neurônios imunorreativos ao Hu. A área do perfil de células gliais entericas S100-IR apresentaram diminuição nos grupos I/R-i e foi detectada proliferação de células gliais entéricas nos grupos I/R-i 0 hora e 24 horas. Conclui-se que a isquemia levou a alterações diferenciadas nos receptores P2X2 e P2X7, células gliais entéricas e neurônios, que podem causar disfunções gastrointestinais.
The nervous system response to various injuries involves the activation of enteric glial cells. The aim of the work was to analyze the effect of ischemia and reperfusion (I/R-i) on enteric glial cells, neurons and receptors P2X2 and P2X7. We analyzed the ileum of Control, Sham and I/R-i with 0 hour, 24 hours and 14 days of reperfusion. Double staining were performed P2X2 and P2X7 receptors with Hu and S100, density, area profile and marking of cellular proliferation. The results show double staining of neurons and enteric glial cell with the P2X2 and P2X7; density increased by glial cells and decrease of neurons immunoreactive to Hu. The area profile of enteric glial cell S100-IR showed decreased in Groups I/R-I and enteric glial cell proliferation was observed in groups I/R-i 0 hours and 24 hours. It is concluded that ischemia has led to changes in differential P2X2 and P2X7 receptors, neurons and enteric glial cells, which can cause gastrointestinal dysfunction.
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42

Mellouk, Amine. "Rôle des récepteurs purinergiques P2X7 et d'apoptose Fas dans l'homéostasie des lymphocytes T et le développement des maladies auto-immunes." Thesis, Université Paris-Saclay (ComUE), 2018. http://www.theses.fr/2018SACLS221.

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Mon étude a porté sur le rôle du récepteur purinergique P2X7 (P2X7R) dans les processus physiopathologiques impliqués dans le développement des maladies auto-immunes de type lupique. Les souris MRL/lpr, déficientes en récepteurs d’apoptose Fas (mutation lpr), développent spontanément ces pathologies suite à l’accumulation lymphocytes T pathogéniques CD4−CD8− (DN) B220+ dans les organes lymphoïdes secondaires. Nous avons observé que ces lymphocytes ont également un déficit d’expression en P2X7R à leur surface. Cela nous a amené à postuler que P2X7R pourrait jouer un rôle clé dans l’homéostasie des lymphocytes T et le développement du lupus. Afin de vérifier notre hypothèse, nous avons produit des souris C57BL/6J (B6) déficientes simultanément pour Fas (lpr) et P2X7R (P2X7KO). Ces souris présentent une accumulation massive de lymphocytes T DN B220+ et des titres très élevés en auto-anticorps et en cytokines proinflammatoires ce qui n’est pas le cas pour les souris B6 simples mutantes lpr ou P2X7KO confirmant pour la première fois l’implication de P2X7R dans l’homéostasie des lymphocytes T, en synergie avec le récepteur Fas. Les lymphocytes T DN pathogéniques responsables de la lymphoaccumulation sont issues majoritairement de la sous populations des lymphocytes T CD8+. L’inflammation chronique présente chez les souris B6/lpr P2X7KO induit l’activation de l’ensemble des populations lymphocytaires T CD4+ et CD8+ naïves conduisant à l’accumulation de lymphocytes T Effecteurs/Mémoires : EM et CM et atteignent parfois le stade exhausted PD1+TIM3+. Ces cellules accumulées CD4+, CD8+ et DN B220+ ont une capacité de réactivation réduite. Ce biais fonctionnel et phénotypique a été confirmé en comparant la réponse immunitaire adaptative anti-adénovirus entre des souris déficientes en Fas et/ou P2X7R. Les réponses cellulaires et humorales sont moins importantes dans les souris B6/lpr P2X7KO que B6, B6-P2X7KO. Ces réponses antivirales sont intermédiaires dans les souris B6/lpr. L’ensemble de ces résultats renforcent notre hypothèse sur le rôle synergique des récepteurs Fas et P2X7R dans l’homéostasie des lymphocytes T. Le taux d’apoptose induit par l’activation des récepteurs Fas ou P2X7R séparément est moins important dans les lymphocytes T CD8+ par rapport au lymphocytes T CD4+. La synergie Fas-P2X7R serait donc nécessaire pour l’homéostasie des lymphocytes T CD8+. Afin de préciser les mécanismes à l’origine de la maladie et d’identifier l’influence de chaque récepteur sur l’expression des loci de susceptibilité, nous avons séquencé les ARNm exprimés dans la rate et les ganglions lymphatiques des souris MRL/lpr avant et après le développement de l’auto-immunité ainsi que chez les souris B6, B6/lpr, B6 P2X7KO et B6/lpr P2X7KO
My project aims to determine the role of the purinergic receptor P2X7 (P2X7R) in the pathophysiological processes involved in the development of autoimmune lupus-like syndrome. MRL/lpr mice, deficient for the cell death receptor Fas (lpr mutation), spontaneously develop this pathology following the accumulation of pathogenic B220+CD4−CD8− (DN) T lymphocytes in secondary lymphoid organs. We have observed that these lymphocytes are also deficient in P2X7R cell surface expression. This led us to hypothesize that P2X7R could play a key role in T cell homeostasis and lupus development. To test our hypothesis, we produced B6 mice deficient for both Fas (lpr) and P2X7R (P2X7KO). These mice, but not single mutant B6 mice (lpr or P2X7KO), develop a massive accumulation of DN B220+ T lymphocytes and high levels of autoantibodies and proinflammatory cytokines, confirming for the first time the involvement of P2X7R in T-cell homeostasis. I have found that the pathogenic DN T lymphocytes are predominantly derived from the CD8+ T lymphocyte subpopulation. Chronic inflammation in B6/lpr P2X7KO mice induces the activation of the whole CD4+ and CD8+ naïve T lymphocyte subpopulations leading to the accumulation of Effector/Memory and exhausted T lymphocytes. Accumulated T-cells lose the ability to be reactivated. To confirm these results, I compared the adaptive immune response against adenovirus between mice deficient for Fas (lpr mutation), P2X7R-deficient mice or both receptors. The cellular and the humoral responses were lower in the B6/lpr-P2X7KO mouse strain compared to B6, B6-P2X7KO and B6/lpr mouse strains. The antiviral immune response in the B6/lpr mice was lower than in B6 and B6-P2X7KO mice. These results reinforce our hypothesis about the synergistic role of both receptors in the maintaining of T cell homeostasis. Ours results suggest that Fas and P2X7R play their synergistic role in T-cell homeostasis. In collaboration with a team from the University of Taiwan, we sequenced the mRNAs expressed in the spleen and lymph nodes of MRL/lpr mice before and after the onset of the diseases as well as in the B6, B6/lpr, B6 P2X7KO and B6/lpr P2X7KO mouse strains in order to better understand the mechanism triggering the disease and to identify the role of each receptor on the expression of the susceptibility loci
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43

Alberto, Anael Viana Pinto. "Caracterização dos receptores P2 em eosinófilos de ratos e do poro associado ao receptor P2X7." Instituto Oswaldo Cruz, 2012. https://www.arca.fiocruz.br/handle/icict/6938.

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Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil
ATP e outros nucleotídeos são liberados para o meio extracelular por vias reguladas ou pela perda da integridade de membrana. Uma vez fora da célula, esses compostos podem ativar receptores P2: P2X (receptores ionotrópicos) e P2Y (receptores acoplados a proteínas G). Além disso, O receptor P2X7 é um importante membro da família P2X, já que sua ativação pode levar a abertura de um poro membranar que permite a passagem de moléculas de até 900 Da. Os eosinófilos são as principais células efetoras em respostas alérgicas e estão associados com diversos processos fisiológicos e patológicos. Nesse trabalho investigamos a expressão de receptores P2 e suas funções em eosinófilos. Nesse contexto, nosso primeiro passo foi investigar a expressão e funcionalidade dos receptores P2X por patch clamping. Nossos resultados sugerem a presença de P2X1, de P2X2 e de P2X7. Em seguida, avaliamos por microfluorimetria a funcionalidade dos receptores P2Y, e verificamos com base na ordem de potência a possível presença de P2Y2, de P2Y4, de P2Y6 e de P2Y11. Além disso, confirmamos nossos dados por imunofluorescência. Realizamos também ensaios de migração in vitro e in vivo, para verificar se os nucleotídeos extracelulares poderiam atrair eosinófilos. O ATP foi capaz de induzir a migração dos eosinófilos, enquanto a suramina, um bloqueador P2, aboliu esse efeito, tanto in vitro, utilizando transwell, como in vivo, utilizando um modelo de pleurisia alérgica em ratos. Em seguida, avaliamos o possível papel da panexina-1 como poro associado ao receptor P2X7. Nesse trabalho utilizamos inibidores de hemicanais em experimentos de patch clamp e em ensaios de permeabilização de corante. Os resultados indicam que os inibidores de hemicanais não bloquearam a geração de corrente ou a captação de corante após a ativação do receptor P2X7 em macrófagos de ratos e camundongos. Demonstramos que eosinófilos de rato expressam receptores P2X e P2Y por imunofluorescência. Além disso, demonstramos que a ativação de receptores P2 pode aumentar a migração de eosinófilos in vitro e in vivo. Além disso, foi demonstrado que inibidores de panexina-1 não bloqueiam a captação do corante ou a corrente gerada pela ativação do receptor P2X7. Os nossos resultados demostraram que panexina-1 não é o poro associado ao receptor P2X7 em macrófagos
ATP and other nucleotides are released from cells through regulated pathways or following the loss of plasma membrane integrity. Once outside the cell, these compounds can activate P2 receptors: P2X ionotropic receptors and G protein-coupled P2Y receptors. . Additionally, P2X7 receptor is an important member of the P2X family of ionotropic receptor as its activation opens a non-selective pore allowing the passage of molecules up to 900 Da. Eosinophils represent major effector cells in the allergic inflammatory response and they are, in fact, associated with several physiological and pathological processes. Here we investigate the expression of P2 receptors and roles of those receptors in murine eosinophils. In this context, our first step were to investigate the expression and functionality of the P2X receptors by patch clamping, our results suggest the presence of P2X1, P2X2 and P2X7. Next we evaluate by microfluorimetry the expression of P2Y receptors, our results based in the ranking order of potency suggests the presence of P2Y2, P2Y4, P2Y6 e P2Y11. Moreover, we confirmed our findings by immunofluorescence assays. We also did in vitro and in vivo migration assays to verify whether nucleotides could attract eosinophil. ATP increased migration of eosinophils, while suramin a P2 blocker abolished this effect in both in vitro, using trasnwell, and in vivo, using a model of rat allergic pleurisy. Next, we evaluated the putative role of pannexin-1 as the pore associated to the P2X7 receptor. We used hemichannels inhibitors in patch clamp and dye uptake experiments. The results indicate that they do not interfere with current generation or dye uptake after activation of P2X7R in rat and mouse macrophages. We have demonstrated that rat eosinophils express P2X and P2Y receptors by immunofluorescence. In addition, the activation of P2 receptors can increase migration of eosinophils in vitro and in vivo. Moreover, we demonstrated that specific inhibitors of pannexin-1 did not interfere with the dye uptake or current generated by the P2X7 activation. Our results showed that pannexin-1 is not the pore associated to the P2X7 receptor in macrophages.
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44

Alqallaf, Sayed Mahmood. "Characterisation of P2X7 receptors in human osteoblasts and modulation by oestrogen." Thesis, Cardiff University, 2008. http://orca.cf.ac.uk/55732/.

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Purinergic P2X7 receptors are ligand-gated ion channels characterized by the formation of a membrane pore upon continuous activation. This receptor has been shown to be expressed and functional in osteoclasts. P2X7 receptor knockout mice studies suggested that this receptor has a role in bone formation. The aim of this project was to characterise the expression and function of P2X7 receptors in human osteoblasts and to investigate the modulation of P2X7 receptor expression and function by oestrogen and glucocorticoids. Reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting were used to study P2X7 receptor expression at the mRNA and protein levels, respectively. P2X7 receptor pharmacology was studied using pore formation and YO-PRO 1 influx in the presence of different agonists and antagonists. The effects of P2X7 receptor activation on physiological aspects of osteoblast function, namely alkaline phosphatase (ALP) production, interleukin-6 (IL-6) and IL-lp release and mineralisation, were also studied. The general findings are: Human osteoblasts (cell lines and primary cells) express functional P2X7 receptors with higher expression seen in primary cells. P2X7 receptor protein is expressed in all the osteoblast-like cell line samples used (lysate, nuclei, and membranes). The functional form of the P2X7 receptor protein appears to correspond to the 67 kDa band and not the 85 kDa band as suggested by the Western blotting results. The pharmacology of P2X7 receptors in human osteoblasts is atypical as evinced by the low affinity of the agonist, benzoyl- benzoyl-adenosine triphosphate (DBzATP) and the weak action of the antagonists, KN62 and brilliant blue-G (BBG). 17p-oestradiol and dexamethasone modulate P2X7 receptor function probably by a non-classical, non-genomic mechanism. ALP production and IL-6 release are increased by P2X7 receptor activation. P2X7 receptor activation appears to decrease mineralisation in primary human osteoblasts. In addition to P2X7 receptors, human osteoblasts express P2X4 and P2X5 receptors. These findings suggest that P2X7 receptors play an important role in osteoblast bone formation and in osteoblast-osteoclast signalling. Targeting P2X7 receptors is a novel approach for the treatment of conditions such as osteoporosis and rheumatoid arthritis. However, further research is needed to develop selective agonists and antagonists.
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45

Lappin, Sarah Crawford. "Biophysical and pharmacological characterisation of recombinant and native rat P2X7 receptors." Thesis, Imperial College London, 2009. http://hdl.handle.net/10044/1/4360.

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P2X7 receptors exhibit a mainly non-neuronal localisation on immune and glial cells and primarily function as non-selective cation channels. After prolonged or repeated exposure to agonist, functional and cellular changes occur: the formation of a large diameter pore, cell lysis and the release of mature, biologically active interleukin-1β (IL-1β) a potent inflammatory cytokine. It is this repertoire of functions, along with its localisation that underlies the hypothesis for its involvement in pain processing. The biophysics and pharmacology of rat P2X7 receptors were investigated using stable cell lines. Increases in the current amplitude were shown to be dependent upon the agonist concentration and current deactivation was agonist application number and voltage dependent. These results increased our understanding of the receptor, but have also had implications for the design of protocols to investigate antagonist potency and efficacy. GSK31418A was identified as a potent, reversible and voltage-independent antagonist of rat and human P2X7 receptors. GSK314181A was >10000 fold selective over P2X4 receptors and >1000 fold selective over P2X2/3 receptors. GSK314181A produced a significant reversal of FCA-induced hypersensitivity when profiled in vivo, providing further validation of the role of P2X7 receptors in inflammatory pain. Although the influence of glia cells on neuronal activity in the CNS is now well documented, the role of peripheral glia, Schwann cells and satellite cells of sensory ganglia, is less well established. Non-neuronal cells in DRG cultures were shown to express P2X7 receptors by pharmacological, biophysical and immunofluorescence techniques. Native P2X7 receptors expressed on these cells were shown to have many of the properties of recombinant P2X7 receptors, in regards to the response to agonist activation and pharmacology. Finally, I have shown that Lamotrigine is an effective inhibitor of recombinant rat and human P2X7 receptors and native P2X7 receptors expressed in DRG. The potent inhibition of human P2X7 by Lamotrigine was replicated with the chemical analogue and neuroprotective agent Sipatrigine. However, little effect was recorded for a P2X7 antagonist in two models of epileptiform activity studies.
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46

Palaskali, Sascha [Verfasser]. "Charakterisierung von Cyclothiazid als Antagonist von humanen P2X7-Rezeptoren / Sascha Palaskali." Ulm : Universität Ulm. Medizinische Fakultät, 2011. http://d-nb.info/1018024883/34.

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47

Hansen, Thomas Sommer. "Targeting inflammation and dysregulated redox signalling via new pathways in cardiovascular disease." Thesis, University of Sydney, 2020. https://hdl.handle.net/2123/24321.

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Background: The role of dysregulated redox signalling and inflammation in driving many cardiovascular disease processes is firmly established. However, therapeutic targeting of redox and inflammatory pathways has been largely unsuccessful to date and there is great scope for improvement with novel therapies. The initial focus of this Thesis was to investigate the role of FXYD1, a protein with known cardiac redox-modulating effects, on pulmonary hypertension (PH). I then focused on the inflammatory pathways in pulmonary hypertension, specifically the NLRP3 inflammasome, using a novel antagonist of the P2X7 receptor (P2X7R)- trifluorinated benzamide 24 (PKT100). For these studies I also developed a non-invasive imaging protocol to allow superior assessment of murine right ventricular function. For the final aims of this Thesis, I sought to establish the therapeutic potential of PKT100 in human monocytes in patients with stable coronary artery disease (CAD) and ST-elevation myocardial infarction (STEMI). I then moved this into murine models of cardiac ischaemia reperfusion and atherosclerosis. Methods and results: Standard C57BL6/J mice were used to establish the protocol for right ventricular (RV) functional assessment. Mice were treated with bleomycin to induce lung fibrosis and secondary PH and increase in right heart afterload. RVs were imaged using high resolution echocardiography with the Vevo 3100 imaging platform. A series of measures were developed and are presented in Chapter 3. This was used in combination with invasive haemodynamics and morphological measurements to identify a role of FXYD1 in protecting against pulmonary vascular remodelling and RV function using FXYD1+/+ and FXYD1-/- mice. FXYD1-/- mice exhibited increased RV pressures, and impaired RV function which were caused by modified redox and inflammatory signalling. A similar protocol was used to assess the therapeutic potential of PKT100 in PH by subcutaneous delivery of the drug to C57BL/6 bleomycin-treated mice. The P2X7R antagonist dramatically improved survival in RV-overloaded mice without improving pulmonary haemodynamics, by an IL-1β-dependent mechanism. I then shifted focus to coronary disease in human patients and extracted peripheral blood mononuclear cells from patients with stable CAD and STEMI. We demonstrated that P2X7R expression in a CD56hiCD16--defined natural killer (NK) cell-subpopulation, was strongly associated with calcium score in multivariate regression analyses. Monocytes were treated ex vivo with PKT100 and inflammatory signalling pathways measured. IL-1β levels were lowered after P2X7R inhibition in cells from STEMI patients with downstream inflammatory activation reduced. Based upon these results we delivered this drug to mice undergoing cardiac ischaemia-reperfusion by reversible coronary artery ligation in 2 cohorts for 60min/24h and 120 min/4wks of ischaemia and reperfusion respectively. Results demonstrated no significant effect on infarct size in the acute cohort, though the chronic cohort showed significant improvements in left ventricular function with PKT100 treatment. We then studied its effects in a mouse model of unstable plaque. ApoE-/- mice were fed a high fat diet, and a tandem stenosis introduced into the right carotid artery at 6 weeks to induce an unstable plaque phenotype. Mice were treated with PKT100 for 7 weeks and plaque development and stability determined by histological analysis of the aortic arch and carotid arteries. P2X7R inhibition attenuated plaque development and exerted demonstrable plaque-stabilising effects. Conclusion: Through the studies presented in this Thesis, I have uncovered two potential therapeutic avenues for pulmonary hypertension, both of which are favourable for addressing the major unmet problem of RV remodelling that leads to fatality. I also found that the novel P2X7R antagonist, developed by our collaborators, is a promising agent for the treatment of both cardiac ischaemia-reperfusion and atherosclerosis. I have patented this drug for use in cardiovascular medicine. This Thesis shows major advancement of a novel pharmacological agent through the early phases of the translational pipeline and future work can build upon this by taking it into further preclinical and clinical studies.
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48

Medhurst, Stephen John. "Investigating the association between P2X7 receptors, microglia and the actions of morphine." Thesis, University of Edinburgh, 2011. http://hdl.handle.net/1842/5539.

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P2X7 receptors belong to a family of membrane bound ion channels which are activated by extracellular ATP, resulting in the opening of a non-selective cation channel. After prolonged or repeated exposure to agonist, functional and cellular changes can occur, including the formation of a large pore, cell lysis and the release of mature, biologically active interleukin-1β. It is this diversity of functions that underlies the significance of this receptor in pain processing. P2X7 receptors are expressed on microglia, which when activated, release a host of mediators which contribute to central sensitisation, a phenomenon associated with neuropathic pain. The role of P2X7 receptors in the activation of microglia is less well established and is the main subject of this thesis. Before considering the interaction between P2X7 receptors and microglia, the first aim was to establish whether P2X7 receptors played a role in a pathological process known to be associated with microglial activation. An additional aim was to establish whether the site of action was in the central nervous system (CNS), where microglia are located. These aims were accomplished using a surgery-based rat model of neuropathic pain, the chronic constriction injury (CCI) model, and by comparing the effects of different P2X7 receptor antagonists when dosed peripherally or directly into the spinal cord. The results indicated that P2X7 receptor antagonists produced efficacy in the CCI model via a mechanism located in the CNS. To further investigate the association between P2X7 receptors and microglia, a different experimental paradigm was explored. Chronically dosed morphine is known to activate microglia, the consequence of which is thought to underlie morphine tolerance and reduced morphine analgesia. By administering a P2X7 receptor antagonist to CCI-operated rats treated with chronic morphine, the interaction between the P2X7 receptor and morphine tolerance and analgesia was explored. The results showed that P2X7 receptor antagonism delayed morphine tolerance and increased the efficacy of low doses of morphine, suggesting an association between P2X7 receptors and microglia. It was intended to confirm the interaction between a P2X7 receptor antagonist and morphine in another neuropathic pain model, namely varicella zoster virus-induced neuropathy. However due to a lack of reproducibility, this model was not used for pharmacological studies. Having demonstrated an association between P2X7 receptor antagonist and morphine in a chronic pain setting, studies were initiated to explore whether this interaction occurred in other morphine-related behaviours. The effect on body weight, motor coordination and single dosed morphine-induced analgesia was assessed in rats co-administered with P2X7 receptor antagonist and morphine. Results demonstrated that the blockade of P2X7 receptors enhanced morphine acute dose-induced analgesia, but had no influence on motor-impairment and body weight. The final part of the thesis used immunohistochemical and molecular techniques to confirm that microglia played a role in established allodynia induced by CCI-surgery and that P2X7 receptors directly influenced microglia-activation. In conclusion, the data in this thesis has illustrated an association between centrally activated P2X7 receptors and microglia, as well as an association between the P2X7 receptor and morphine-induced tolerance and analgesia. It is possible that co-administration of a P2X7 receptor antagonist with morphine could reduce the effective dose of morphine clinically, thereby reducing the side effects of this commonly used analgesic.
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Santos, Junior André Avelino dos. "Papel do receptor P2X7 em modelo murino de infecção por Mycobacterium tuberculosis." Pontifícia Universidade Católica do Rio Grande do Sul, 2012. http://hdl.handle.net/10923/1395.

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Tuberculosis (TB) remains a leading cause of death worldwide, due to the great adaptability of the bacillus, which can survive in various conditions inside and outside the human host. Previous studies showed evidence that polymorphisms in P2X7 receptor are e associated with increased risk of TB. The present study aimed to analyze the role of purinergic P2X7 receptor in M. tuberculosis infection and host interaction mechanisms in vitro and in vivo. The macrophage murine cell line RAW 264. 7 was used for in vitro experiments. Our results demonstrated that treatment of RAW 264. 7 cells with ATP (3 and 5 mM) resulted in a statistically significant reduction of counting colony forming units (CFUs). Male wild-type C57BL/6 (wild-type) and P2X7 receptor KO (P2X7R-/-) mice (25–30 g) were used throughout this study for in vivo. Immunohistochemistry showed that the purinergic P2X7 receptor expression was found significantly augmented in the lungs of mice infected with M. tuberculosis. Furthermore, M. tuberculosis-infected P2X7R-/- mice showed an increase of M. tuberculosis burden in lung tissue, when compared to infected wild type mice. Infected mice showed a marked increase in the spleen weight, in comparison to non-infected animals, indicating the occurrence of splenomegaly. In P2X7R-/- spleens, we observed a significant decrease in the populations of Treg (CD4+Foxp3+), T cells (CD4+, CD8+CD25+ and CD4+CD25+), dendritic cells (CD11c+) and B220+ cells. However, a significant increase in CD11b+ cells was observed in P2X7R-/- mice, when compared to wild-type animals. In the lungs, P2X7R-/- M. tuberculosis-infected mice exhibited pulmonary infiltrates containing an increase of Treg cells (CD4+Foxp3+), T cells (CD4+ and CD8+) and a decrease in the B220+ cells, when compared with wild-type M. tuberculosis-infected mice. The findings observed in the present study provide novel evidence on the role of P2X7 receptors in the pathogenesis and control of tuberculosis. Whether selective agonists or antagonists of this receptor might be useful for improving TB complications remains a matter to be investigated.
A tuberculose (TB) continua sendo uma das principais causas de morte no mundo, devido à grande capacidade de adaptação do bacilo que pode sobreviver em diferentes condições dentro e fora do hospedeiro humano. Estudos prévios mostraram evidências de que polimorfismos no receptor purinérgico P2X7 estão associados ao aumento da suscetibilidade à TB. O presente estudo objetivou analisar o papel do receptor purinérgico P2X7, na infecção por M. tuberculosis em camundongos, e os possíveis mecanismos de interação do receptor P2X7 com o hospedeiro, em modelos in vivo e in vitro. Nos experimentos para avaliação da viabilidade da micobacteria intracelular in vitro foi utilizada a linhagem de macrófagos murinos, RAW 264. 7. Nossos resultados demonstraram que o tratamento das células RAW 264. 7 com ATP (3 e 5 mM) resultou em uma redução estatisticamente significativa da contagem de unidades formadoras de colônias (CFUs). Nos experimentos in vivo foram utilizados camundongos machos C57BL/6 (tipo selvagem) e camundongos knockout para o receptor P2X7 (P2X7R-/-) (25-30 g). Os resultados com imuno-histoquímica mostraram que a expressão do receptor purinérgico P2X7 foi encontrada significativamente aumentada nos pulmões de camundongos infectados com M. tuberculosis. Além disso, camundongos P2X7R-/- infectados com M. tuberculosis mostraram um aumento da carga da micobacteria no tecido pulmonar, quando comparado com camundongos do tipo selvagem infectados. Camundongos infectados mostraram um aumento marcante no peso do baço quando comparado com animais não infectados, indicando a ocorrência de esplenomegalia. Em baços de camundongos P2X7R-/-, observou-se uma diminuição significativa nas populações de Treg (CD4 + Foxp3 +), células T (CD4 +, CD8 + CD25 + e CD4 + CD25 +), células dendríticas (CD11c +) e células + B220. No entanto, houve um aumento significativo de células CD11b + em camundongos P2X7R-/-, quando comparados aos animais do tipo selvagem. Nos pulmões, camundongos P2X7R-/- infectados com M. tuberculosis apresentaram infiltrado pulmonar contendo um aumento de células Treg (CD4 + Foxp3 +), células T (CD4 + e CD8 +) e uma diminuição nas células + B220, quando comparado com camundongos do tipo selvagem infectados com M. tuberculosis. Portanto, os resultados observados no presente estudo fornecem novas evidências sobre o papel dos receptores P2X7 na patogênese e controle da tuberculose. O uso de agonistas ou antagonistas seletivos deste receptor como uma ferramenta terapêutica continua sendo uma questão a ser investigada.
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50

SALARO, ERICA. "Involvement of the P2X7-NLRP3 axisi in Leukemic Cell Proliferation and Death." Doctoral thesis, Università degli studi di Ferrara, 2016. http://hdl.handle.net/11392/2403212.

Full text
Abstract:
Lymphocyte growth and differentiation are modulated by extracellular nucleotides and P2 receptors. We previously showed that the P2X7 receptor (P2X7R or P2RX7) is overexpressed in circulating lymphocytes from chronic lymphocytic leukemia (CLL) patients. In the present study we investigated the P2X7R/NLRP3 inflammasome axis in lymphocytes from a cohort of 23 CLL patients. P2X7R was overexpressed and correlated with chromosome 12 trisomy in CLL patients. ASC mRNA and protein were also overexpressed. On the contrary, NLRP3 were dramatically down-modulated in CLL lymphocytes relative to lymphocytes from healthy donors. To further investigate the correlation between P2X7R, NLRP3 and cell growth, NLRP3 was silenced in THP-1 cells, a leukemic cell line that natively expresses both NLRP3 and P2X7R. NLRP3 silencing enhanced P2X7R expression and promoted growth. On the contrary, NLRP3 overexpression caused accelerated apoptosis. In conclusion, we show NLRP3 down-modulation stimulates P2X7R expression and promotes growth, while NLRP3 overexpression inhibits cell proliferation and stimulates apoptosis. These findings suggest that NLRP3 is a negative regulator of growth and point to a role of the P2X7R/NLRP3 axis in CLL.
La crescita ed il differenziamento dei linfociti sono modulati da nucleotidi extracellulari e dai recettori P2. Abbiamo precedentemente dimostrato che il recettore P2X7 (P2X7R o P2RX7) è sovraespresso nei linfociti circolanti provenienti da pazienti affetti da Leucemia Linfatica Cronica (LLC). In questo studio abbiamo valutato l’asse P2X7RR/NLRP3 inflammasoma nei linfociti da una coorte di 23 pazienti affetti da LLC. Abbiamo visto che P2X7R era sovra espresso e correla con la trisomia del cromosoma 12 in pazienti affetti da LLC. Allo stesso modo mRNA e proteine di ASC sono sovraespresse. Al contrario, NLRP3 è drasticamente sotto modulato nei linfociti dei pazienti di LLC comparati ai linfociti isolati da donatori sani. Per meglio approfondire la correlazione tra P2X7R, NLRP3 e la crescita cellulare, NLRP3 è stato silenziato in cellule THP-1, una linea cellulare leucemica che nativamente esprime sia NLRP3 che P2X7R. Il silenziamneto di NLRP3 aumenta l’espressione di P2X7R e favorisce la crescita. Al contrario, la sovra espressione di NLRP3 causa un accelerazione del processo apoptotico. In conclusione, abbiamo visto che la sotto espressione di NLRP3 stimula l’espressione di P2X7R e favorisce la crescita, mentre la sovra espressione di NLRP3 inibisce la proliferazione cellulare e stimola l’apoptosi. Questi risultati suggeriscono che NLRP3 è un regolatore negativo della crescita e che l’asse P2X7R/NLRP3 può avere un ruolo importante nella LLC.
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