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Schiavuzzo, Jalile Garcia 1980. "Mecanismos envolvidos na ação hiperalgésica induzida pela ativação de receptores P2X3 e P2X2/3 no músculo gastrocnêmio de ratos." [s.n.], 2013. http://repositorio.unicamp.br/jspui/handle/REPOSIP/244518.
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Orientador: Maria Cláudia Gonçalves de Oliveira FusaroDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Aplicadas
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Resumo: Existem evidências do envolvimento do ATP via ativação do receptor P2X3 na dor muscular. Portanto, o objetivo deste estudo foi verificar se a ativação do receptor P2X3 no músculo gastrocnêmio de ratos induz hiperalgesia mecânica, e em caso afirmativo, analisar os mecanismos inflamatórios pelo qual os receptores P2x3 induzem hiperalgesia mecânica. O Antagonista não seletivo para o receptor P2X3 ?,?meATP foi administrado no músculo gastrocnêmio de ratos, induzindo hiperalgesia, a qual foi significativamente reduzida pelo antagonista seletivo do receptor P2X3 e P2X2/3 - A-317491. A hiperalgesia mecânica induzida pelo ?,?meATP foi reduzida pelo inibidor de ciclooxigenase Indometacina, pelo antagonista seletivo do receptor de Bradicinina B1 e B2- Dalbk e Bradyzide, respectivamente, antagonista dos adrenoceptores ?1 e ?2 - Atenolol e ICI 118,551 respectivamente, e inibidor não específico de selectinas Fucoidan. O ?,?meATP também induziu o aumento da concentração local de citocinas pro inflamatórias TNF-?, IL-1?, IL-6 e CIN e migração de neutrófilos. Juntos estes achados sugerem que o ?,?meATP induz hiperalgesia mecânica no músculo gastrocnêmio via ativação de receptor periférico P2X3, o qual envolve bradicinina, prostaglandinas e aminas simpatomiméticas e migração de neutrófilos. Portanto, nós sugerimos que os receptores P2X3 sejam um importante alvo no controle da dor muscular
Abstract: There is evidence of the involvement of endogenous ATP via activation of P2X3 in muscle pain. Therefore, the aim of this study was to verify whether the activation of P2X3 receptors in the gastrocnêmio muscle of rats induces mechanical hyperalgesia and, if so, to analyze the inflammatory mechanisms by which P2X3 receptors induce mechanical hyperalgesia. Intramuscular administration of the non-selective P2X3 receptor agonist ?,?-meATP in the gastrocnemius muscle of rats induced mechanical hyperalgesia, which was significantly reduced by the selective P2X3 and P2X2/3 receptors antagonist A-317491. The ?,?-meATP-induced mechanical hyperalgesia was prevented by the indomethacin cyclooxygenase inhibitor, the selective bradykinin B1- or B2- receptor antagonist DALBK and bradyzide, respectively, the ?1- or ?2-adrenoceptor antagonist atenolol and ICI 118,551, respectively, and the nonspecific selectin inhibitor fucoidan. ?,?-meATP also induced increase in the local concentration of the pro-inflammatory cytokines TNF-?, IL-1?, IL-6 and CINC-1 and the neutrophil migration. Together, these findings suggest that ?,?-meATP induced mechanical VIII hyperalgesia in the gastrocnemius muscle of rats via activation of peripheral P2X3 receptors, which involves bradykinin, prostaglandins, sympathetic amines, pro-inflammatory cytokines and neutrophil migration. Therefore, we suggest that P2X3 receptors are important targets to control muscle inflammatory pain
Mestrado
Biodinâmica do Movimento Humano e Esporte
Mestra em Ciências da Nutrição e do Esporte e Metabolismo
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Chabot-Doré, Anne-Julie. "Metabotropic regulation of ATP-gated P2X3 receptors." Thesis, McGill University, 2006. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=101708.
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Among the ATP-gated channels, the P2X3 subtype is exclusively expressed in nociceptors of dorsal root ganglia (DRG) and trigeminal ganglia, where it plays a major role in enhanced pain sensation observed in chronic pain states. We tested the hypothesis that P2X3 receptors are modulated by metabotropic receptors, such as 5-HT2A, mG1uR5 and trkA, leading to increased P2X3-mediated currents. Double fluorescence labeling confirmed that P2X3-expressing neurons are labeled by the lectin IB4 and we showed that 5-HT2A and mGluR5 receptors, but not trkA, are expressed in a fraction of IB4-positive neurons. Using confocal microscopy, we examined the subcellular distribution of P2X3 and we observed that 5-HT induced a translocation of P2X3 labeling in a significant number of neurons. In Xenopus oocytes, we recorded a short-lasting and kinase-dependent potentiation of P2X3 currents by activation of co-expressed 5-HT2A and mGluR5 receptors. The data presented here show that both 5-HT2A and mG1uR5 are potential modulators of P2X3 receptors in a subset of nociceptors in DRG.
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Wang, Haihong. "Amino acid residues constituting the agonist binding site of the human P2X3 receptor and subunit stoichiometry of heteromeric P2X2/3 and P2X2/6 receptors." Doctoral thesis, Universitätsbibliothek Leipzig, 2013. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-112913.
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Homotrimeric P2X3 and heteromeric P2X2/3 receptors are present in sensory ganglia and participate in pain perception. In order to develop pharmacological antagonists for these receptors, it is important to clarify which amino acid (AA) residues constitute the agonist binding pouch as well as to learn the stoichiometry of the receptor subunits forming a heteromeric receptor. We expressed the homomeric human (h)P2X3 receptor or its mutants in HEK293 cells and measured the ATP-induced responses by the whole-cell patch-clamp method. For the binding-site mutants, all conserved and some non-conserved AAs in the four nucleotide binding segments (NBSs) of the P2X3 subunit were sequentially replaced by alanine. Especially the positively charged AAs Lys and Arg appeared to be of critical importance for the agonist effects. We concluded that groups of AAs organized in NBSs rather than individual amino acids appear to be responsible for agonist binding at the P2X3 receptor. These NBSs are located at the interface of the three subunits forming a functional receptor. We were also interested to find out, whether two heteromeric receptors (P2X2/3 and P2X2/6), where P2X2 combines with two different partners, have an obligatory subunit stoichiometry of 1:2 or whether the subunit stoichiometry may be variable. For this purpose we used non-functional P2X2, P2X3 and P2X6 subunit-mutants to investigate the composition of heteromeric P2X2/3 and P2X2/6 receptors. The subunit stoichiometry of P2X2/3 and P2X2/6 was found to be 1:2 and 2:1, respectively. Thus, recognitions sites between P2X2 and its partners rather than random association may govern the subunit compositions of the receptor trimers.
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Mo, Gary. "Molecular physiology of sensory P2X3 ATP receptor channels." Thesis, McGill University, 2012. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=107793.
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Purinergic transmission mediated by extracellular release of ATP has been shown to be involved in numerous physiological processes, ranging from bladder function to taste and hearing. The diverse roles of ATP mediated signaling is largely due to widespread distribution of P2 ATP receptors. Since their initial cloning, various P2 receptors have been found to mediate a wide range of cellular processes in different tissues. One P2 receptor in particular, the P2X3 receptor, is almost exclusively expressed on nociceptive dorsal root ganglion (DRG) sensory neurons. Due to its specific distribution, the P2X3 receptor has been a prominent target in pain research, especially in studies of chronic pain conditions. Numerous studies have indicated involvement of P2X3 receptor in mediating increased pain behavior associated with chronic inflammation or neuropathic injury. However, the exact contribution of the P2X3 in chronic pain conditions is still unclear, especially in the case of neuropathic pain. There is inconsistency in reports of changes in P2X3 expression or activity during neuropathic pain. The underlying element of enhanced pain behavior after nerve injury is increased excitability of sensory neurons. The first study of described in this thesis investigates the contribution of the P2X3 receptor to changes in excitability of injured neurons. Activation of protein kinase C (PKC) has been shown to facilitate chronic pain behavior by modulating the activity of various ion channels. Thus, the contribution of PKC to hyperexcitability in injured neurons was also investigated in the first study.Nerve injury induces very dynamic changes in cellular physiology including activation of various intracellular signaling pathways. The activity of the P2X3 receptor may be affected by such cellular processes. Understanding the molecular physiology of the P2X3 receptor may provide additional insight into the specific contribution of the P2X3 receptor in pain physiology. A common component of many cellular signaling pathways is the cleavage of phosphatidylinositol 4,5-bisphosphate (PIP2) by phospholipase C (PLC), and changes in PIP2 levels modulate the activity of various ion channels, including the P2X2 receptor. The second study in this thesis investigates the modulation of P2X3 by changes in intracellular levels of PIP2. Recent studies have demonstrated possible co-expression of metabotropic P2Y receptors on P2X3 positive sensory neurons. ATP release on sensory neurons that express both P2Y and P2X3 receptors will likely trigger activation of both types of purinergic receptors. The third study in this thesis investigated the modulation of P2X3 receptor activity by metabotropic P2Y receptors. Our understanding of the P2X3 receptor predominantly comes from rodent studies. Because interspecies differences in the functional characteristics of certain P2X receptor subtypes have been reported, there is a pressing need to verify if our understanding of the rodent P2X3 receptor is translatable to species closer to man. To this end, the last study of this thesis compares the pharmacological properties of native P2X3 receptor on rat sensory neurons to native P2X3 receptors on monkey sensory neurons.Il a été montré que la transmission purinergique médiée par la libération extracellulaire d'ATP est impliquée dans de nombreux processus physiologiques, allant du fonctionnement de la vessie aux sens du goût et de l'audition. Les rôles variés de la signalisation par l'ATP sont expliqués principalement par la distribution étendue des récepteurs P2 de l'ATP dans l'organisme. Depuis leur clonage initial dans les années '90, une variété de récepteurs P2 régissant divers mécanismes cellulaires a été découverte dans plusieurs tissus. Un récepteur canal P2 en particulier, P2X3, se trouve exprimé quasi-exclusivement dans les neurones nocicepteurs des ganglions spinaux (DRG). De par cette distribution spécifique, le récepteur P2X3 est une cible importante dans la recherche sur la douleur, principalement dans les études sur la douleur chronique. De nombreuses études indiquent le rôle du récepteur P2X3 dans l'augmentation des réponses à la douleur associée à une inflammation chronique ou une lésion neuropathique. Cependant, la contribution exacte de P2X3 dans la douleur chronique reste incertaine, surtout dans les cas de douleur neuropathqiue. Il existe des contradictions dans les articles sur les changements d'expression ou d'activité de P2X3 en conditions de douleur neuropathique. Un élément clé dans l'exacerbation des comportements douloureux après lésion neuropathique est l'augmentation d'excitabilité des neurones sensoriels. La première étude décrite dans cette thèse explore la contribution de P2X3 dans les changements d'excitabilité des neurones de DRG endommagé. Il a été rapporté que l'activation de la protéine kinase C (PKC) facilite les comportements douloureux en modulant l'activité de certains canaux ioniques. Ainsi, la contribution de PKC à l'hyperexcitabilité des neurones de DRG neuropathique a aussi été étudiée dans ce premier chapitre. Une insulte à un nerf périphérique induit des changements très dynamiques dans sa physiologie cellulaire, incluant l'activation de voies de signalisation intracellulaires. La fonction de P2X3 peut se trouver affectée par ces mécanismes neuronaux. Comprendre la physiologie moléculaire du récepteur P2X3 peut nous éclairer sur sa contribution spécifique dans la douleur. Une étape commune à de nombreuses voies de signalisation cellulaire est le clivage du phosphatidylinositol 4,5-bisphosphate (PIP2) par la phospholipase C (PLC). Les variations de niveaux de PIP2 modulent l'activité de plusieurs canaux ioniques, y compris le récepteur P2X2. Le deuxième chapitre dans cette thèse se concentre sur la modulation de P2X3 par les niveaux intracellulaires de PIP2. Des études récentes ont démontré la co-expression potentielle de récepteurs métabotropiques P2Y et ionotropiques P2X3 sur les neurones sensoriels. L'ATP pouvant activer les deux types de récepteurs, le troisième chapitre se penche sur la modulation de la fonction de P2X3 par les récepteurs P2Y couplés à la phospholipase C.Notre compréhension du récepteur P2X3 provient principalement des données obtenues dans des modèles précliniques de rongeurs. Sachant que des différences interspécifiques marquées dans les propriétés fonctionnelles de certains sous-types de récepteurs P2X ont été documentées, il est urgent et important de vérifier que nos connaissances sur le récepteur P2X3 de rongeur sont transférables aux primates ou à l'homme. À cette fin, dans la dernière étude de cette thèse, nous comparons les propriétés pharmacologiques du récepteur P2X3 natif à la surface des neurones sensoriels de rat avec celles du récepteur P2X3 exprimé dans les neurones sensoriels de singe.
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Günther, Janka [Verfasser]. "Generierung und Charakterisierung transgener BAC-P2X3-Mäuse / Janka Günther." Aachen : Hochschulbibliothek der Rheinisch-Westfälischen Technischen Hochschule Aachen, 2014. http://d-nb.info/1051414172/34.
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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 TambeliTese (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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Rashed, Mahmoud [Verfasser]. "Syntheses and structure-activity relationships of novel P2X3 receptor antagonists / Mahmoud Rashed." Bonn : Universitäts- und Landesbibliothek Bonn, 2018. http://d-nb.info/1160594392/34.
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Prado, Filipe César do. "Papel do receptor P2X3 e da ativação da proteína kinase C épsilon dos neurônios nociceptivos periféricos na dor inflamatória." [s.n.], 2010. http://repositorio.unicamp.br/jspui/handle/REPOSIP/314727.
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Orientador: Carlos Amílcar ParadaDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: Enquanto a hiperalgesia inflamatória depende da liberação de prostaglandinas e/ou de aminas simpatomiméticas que sensibilizam os neurônios aferentes primários, nosso grupo demonstrou recentemente que o bloqueio do receptor P2X3 no tecido periférico previne a hiperalgesia induzida pela carragenina.. No entanto, o mecanismo pelo qual a ativação dos receptores P2X3 neuronais contribui para a hiperalgesia inflamatória não está completamente estabelecido. O presente estudo verifica se a ativação do receptor P2X3 dos neurônios aferentes primários contribui para a hiperalgesia mecânica induzida pela prostaglandina E2 ou pela dopamine no tecido periférico. A co-administração de A317491 (60 µg / paw), um antagonista seletivo do receptor P2X3, ou o prétratamento com dexametasona (1 mg / mL / kg), preveniu a hiperalgesia mecânica medida 3 horas depois da administraçao de carragenina (300 µg / paw) na pata posterior de ratos. A administração de ??meATP (50 µg /paw) induziu hiperalgesia mecânica 1 hora, mas não 3 horas, depois da sua administração, que foi prevenida pela dexametasona ou pelo A317491. Doses sublimiares de PGE2 (4 ng / paw) ou dopamina (0.4 µg / paw) que não induzem hiperalgesia por si só, induziram hiperalgesia, 3 horas depois, quando administradas logo depois de ??meATP ou carragenina em ratos tratados com dexametasona. Esses estados de hiperalgesia ("priming") revelados pelas doses sublimiares de PGE2 ou dopamine foram prevenidos pelo A317491 ou pelo tratamento com administração intraganglionar (DRG-L5) de ODN antisense, mas não pelo ODN mismatch, contra o receptor P2X3 (40 µg /5µL once a day for 4 days). ODN antisense, mas não o ODN mismatch, reduziu a expressão dos receptores P2X3 no nervo safeno e no DRG-L5. Para verificar se a PKC? media esse estado de hiperalgesia, inibidor de translocação de PKC? (1 µg/paw) foi administrado no tecido periférico 45 minutos antes do ??meATP ou PGE2 (100 ng/paw). O inibidor de PKC? preveniu o estado de hiperalgesia induzido pelo ??meATP ("priming"), mas não a hiperalgesia mecânica induzida pela PGE2 (100 ng/paw). Dessa maneira, os resultados desse estudo sugerem que a hiperalgesia inflamatória depended a ativação dos receptores P2X3 neuronais e da subsequente translocação da PKC? , que aumenta a susceptibilidade dos neurônios aferentes primários (priming) à ação de outros mediadores inflamatórios como a PGE2 e as aminas simpatomiméticas
Abstract: While inflammatory hyperalgesia depends on the release of prostaglandins and/or sympathetic amines that ultimately sensitize the primary afferent neurons, we have recently demonstrated that blockade of P2X3 receptor in the peripheral tissue completely prevents carrageenan-induced hyperalgesia. However, the mechanism by which the activation of neuronal P2X3 receptor contributes to the inflammatory hyperalgesia is not completely clear. The present study verifies whether the activation of P2X3 receptor on primary afferent neurons contributes to the mechanical hiperalgesia induced by prostaglandin E2 or dopamine in the peripheral tissue. Co-administration of A317491(60 µg / paw), a selective P2X3,2/3 receptor antagonist, or pre-treatment with dexamethasone (1 mg / mL / Kg), prevented the mechanical hyperalgesia measured 3 hours after the administration of carrageenan (300 µg / paw) in the rat's hind paw. The administration of ??meATP (50 µg /paw) induced mechanical hiperalgesia 1 hour, but not 3 hours, after its administration, which also was prevented by dexamethasone or A317491. Sub-threshold doses of PGE2 (4 ng / paw) or dopamine (0.4 µg / paw) that do not induce hyperalgesia by themselves, induced maximal hyperalgesia, 3 hours after, when administrated Just following ??meATP or carrageenan in rats treated with dexamethasone. These hyperalgesic states ("priming") revealed by sub-threshold doses of PGE2 or dopamine were prevented by A317491 or treatment with ganglionar administrations (DRG-L5) of ODN antisense, but not ODN mismatch, against P2X3 receptor (40 µg /5µL once a day for 4 days). ODN antisense, but not ODN mismatch reduced the expression of P2X3 receptors in the saphenous nerve and in DRG-L5. To verify whether PKC? mediates this hyperalgesic state, PKC? translocation inhibitor (1 µg/paw) was administrated in peripheral tissue 45 min. before ??meATP or PGE2 (100 ng/paw). PKC? inhibitor inhibited the hyperalgesic state induced by ??meATP ("priming"), but not the mechanical hyperalgesia induced by PGE2 (100 ng/paw). Briefly, the findings of this study suggest that the inflammatory hyperalgesia depends on neuronal activation of P2X3 receptor and the subsequent PKC? translocation, which increases the susceptibility of primary afferent neurons (priming) to others inflammatory mediators such as PGE2 and symphatetic amines
Mestrado
Fisiologia
Mestre em Biologia Funcional e Molecular
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Chen, Zhicheng. "Molecular cloning and characterisation of a sensory neuron-specific ATP-gated channel (P2X3)." Thesis, University College London (University of London), 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.285158.
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Helms, Nick. "Wechselwirkungen von Agonisten und kompetitiven Antagonisten mit der Ligandenbindungsstelle des schnell desensitisierenden P2X3-Rezeptors." Doctoral thesis, Universitätsbibliothek Leipzig, 2016. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-197364.
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Purinerge P2X3-Rezeptoren spielen eine bedeutende Rolle in der Vermittlung chronischer Schmerzen, welche ein führendes Problem des Gesundheitswesens mit vielen sozioökonomischen Konsequenzen darstellen. Die Tatsache, dass P2X3-Rezeptoren fast ausschließlich von
nozizeptiven Neuronen exprimiert werden, macht sie trotz ihres besonderen Desensitisierungsverhaltens zu vielversprechenden Angriffspunkten zukünftiger Schmerztherapien, beispielsweise mithilfe kompetitiver Antagonisten an diesen Rezeptoren. Zur Analyse der Wechselwirkungen zwischen Agonist und kompetitivem Antagonist wird meist der Schild-Plot benutzt. Jedoch ist dieser im Falle der sehr schnell desensitisierenden P2X3-Rezeptoren ungeeignet, da die Vorbedingung
eines stabilen Gleichgewichts zwischen Agonist und Antagonist aufgrund der Desensitisierung nicht erfüllt ist.
Ziel der vorliegenden Arbeit war es, eine neue Methode zur Analyse der Interaktion kompetitiver Antagonisten mit ihrer Bindungsstelle am Beispiel des P2X3-Rezeptors zu entwickeln und so für die Antagonistenbindung bedeutende Aminosäuren der Bindungsstelle zu identifizieren.
Mittels der Patch-Clamp-Technik wurden die Effekte der Antagonisten A-317491, TNP-ATP und PPADS auf die vom P2X1,3-Rezeptor-selektiven Agonisten α,β-MeATP induzierten Ströme am P2X3-Wildtyp-Rezeptor und an fünf Rezeptormutanten mit veränderter Ligandenbindungsstelle
untersucht. Alle Rezeptoren wurden in HEK293-Zellen exprimiert. Anhand der gemessenen Daten wurde ein Hidden Markov Model (HMM) erstellt, welches die sequentiellen Übergänge des Rezeptors von geschlossen zu offen und desensitisiert in An- und Abwesenheit des Antagonisten miteinander kombiniert. Die am P2X3-Rezeptor induzierten Ströme konnten mithilfe dieses Modells korrekt gefittet und die für die Antagonistenbindung wichtigen Aminosäuren innerhalb der Bindungsstelle bestimmt werden. Als Resultat dieser Arbeit konnte außerdem gezeigt werden, dass das HMM eine geeignete Methode zur Analyse der Wirkung kompetitiver Antagonisten an schnell desensitisierenden Rezeptoren darstellt. Die untersuchten Antagonisten A-317491 und TNP-ATP haben einen kompetitiven Wirkmechanismus, während PPADS eine pseudoirreversible Blockade verursacht.
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Wang, Haihong [Verfasser], Peter [Akademischer Betreuer] Illes, Wolfgang [Gutachter] Nörenberg, and Andreas [Gutachter] Reichenbach. "Amino acid residues constituting the agonist binding site of the human P2X3 receptor and subunit stoichiometry of heteromeric P2X2/3 and P2X2/6 receptors / Haihong Wang ; Gutachter: Wolfgang Nörenberg, Andreas Reichenbach ; Betreuer: Peter Illes." Leipzig : Universitätsbibliothek Leipzig, 2013. http://d-nb.info/1238366546/34.
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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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Majumder, Paromita. "Análise dos receptores P2X2 e P2X4 durante a diferenciação neuronal." Universidade de São Paulo, 2007. http://www.teses.usp.br/teses/disponiveis/46/46131/tde-05102007-145008/.
Full textAbstract:
Durante o desenvolvimento do sistema nervoso, as oscilações da concentração de cálcio intracelular livre resultam na proliferação celular, migração e diferenciação neuronal. Nesta tese foram investigadas a participação dos receptores ionotrópicos purinérgicos dos tipos P2X2 e P2X4 seletivos ao influxo de cálcio durante a diferenciação neuronal in vitro das células de carcinoma embrionário murino P19. Identificamos o padrão diferencial de expressão de receptores purinérgicos nas células indiferenciadas e neurônios P19. O receptor P2X4 é expresso durante toda a diferenciação neuronal e o receptor P2X2 é detectado na fase tardia da diferenciação em neurônios. Através de ensaios farmacológicos, foi possível identificar a participação dos receptores metabotropicos P2Y e do receptor P2X4 na formação dos corpos embriônicos, na proliferação celular e ou na determinação do fenótipo de progenitor neural. Durante a maturação neuronal os receptores P2X2 e P2Y1 participam da determinação do fenótipo neuronal glutamatérgico NMDA e os receptores P2X2 e P2Y2 no fenótipo neuronal colinérgico. A ausência de inibidores específicos e seletivos aos receptores purinérgicos levou-nos a empregar a técnica SELEX (Systematic Evolution of Ligands by EXponential enrichment) a fim de identificar inibidores seletivos aos receptores P2X2 e P2X4. A técnica envolve a utilização da biblioteca combinatória randômica de RNA 2\'- F pirimidina modificadas resistentes a nucleases. Após 9 ciclos de seleção in vitro de SELEX (ciclo 9-P2X4), as sequências selecionadas mostraram-se seletivas a ligação somente ao receptor P2X4 e não aos receptores P2X2 ou P2X7 através de ensaios de ligação radioligante-receptor. Por patch clamping na configuração whole cell recording identificou-se que além de seletividade ao receptor, que a aplicação do RNA ciclo 9- P2X4 promoveu inibição da corrente ativada pelo ATP somente nos receptores P2X4 e não em P2X2 em celulas 1321N1 astrocitoma transfectadas. A incubação do RNA ciclo 9-P2X4 na concentração de 200 nM com as células no estágio indiferenciado inibiu a formação dos corpos embriônicos. Já utilização de 25 nM, resultou em mudanças morfológicas nas células diferenciadas. Estes dados corroboram com os dados farmacológicos que identificaram a participação do receptor P2X4 na diferenciação precoce. Após 11 ciclos P2X2 de seleção, identificou-se sequências com especificidade de ligação aos receptores P2X2. Aptâmeros, moleculas de RNA com sequência identificada e com alta afinidade ao alvo da seleção, foram isolados de ambas as bibliotecas, ciclo 9 P2X4 e ciclo 11 P2X2. A co-aplicação destes aptâmeros e ATP em ensaios de whole-cell recording resultou na inibição de 30 a 80% da corrente ativada pelo ATP nos receptores P2X2 ou P2X4. Estes testes em células PC12 de rato, que expressa os receptores endógenos, resultou em inibição da corrente ativada pelo ATP de modo semelhante. Além de termos desenvolvido aptâmeros como ferramentas para elucidar as funções dos receptores P2X2 e P2X4 durante o desenvolvimento, diferenciação, em processos fisiológicos e patológicos, estas moléculas resistentes a nucleases são as primeiras identificadas capazes de reconhecer, discernir e inibir dois subtipos de receptores purinérgicos sendo promissores para utilização terapêutica.During the development of the nervous system, oscillations of intracellular calcium concentrations activate programs of gene expression resulting in proliferation, migration and neuronal differentiation of embryonic cells. In this thesis, the participation of ionotropic P2X2 and P2X4 receptor subtypes, whose receptor channels are highly permeable for calcium influx in the cells, was studied during the process of neuronal differentiation. We have identified differential gene expression of purinergic receptors in undifferentiated and neuronal-differentiated P19 cells. P2X4 receptor expression was present along neuronal differentiation of P19 cells, whereas P2X2 receptor expression was only detected when P19 cells became neurons. Based on purinergic receptor pharmacology we have determined the participation of P2X4 receptors in addition to metabotropic P2Y2 receptors in the formation of embryonic bodies as prerequisites for phenotype determination of P19 neural progenitor cells. Final neuronal maturation of P19 cells in the presence or absence of agonists or antagonists of purinergic receptors implicated the involvement of P2X2, P2Y1, and P2Y2 in the determination of the final neuronal phenotype, such as expression of NMDA-glutamate and cholinergic receptors. In order to further evaluate the functions of these P2X receptors and due to the absence of specific inhibitors for these receptor subtypes, we have used the SELEX technique (Systematic Evolution of Ligands by EXponential enrichment) to select for specific inhibitors for P2X2 and P2X4 receptors. The 2\' -F-pyrimidine modified, nuclease- resistant combinatorial SELEX RNA pool enriched with inhibitors of P2X4 receptors following nine cycles of in vitro selection (cycle 9-P2X4) specifically interacted with P2X4 receptors and not with P2X2 or P2X7 receptors as verified in radioligand-receptor binding studies. Moreover, whole-cell recording measurements using astrocytoma cells expressing recombinant rat P2X2 or P2X4 receptors showed inhibition of P2X4 but not of P2X2 receptors by the selected RNA molecules. RNA molecules selected in vitro in 11 reiterative SELEX cycles using the P2X2 receptor as target specifically bound to membrane extracts containing recombinant P2X2 receptors. From both selected RNA libraries (against P2X4 and P2X2 receptors) aptamers, as RNA molecules with identified sequences and high-affinity binding, were identified by cloning and DNA sequencing. The presence of these aptamers in whole-cell recording experiments resulted in 30-80% inhibition of ATP-induced receptor activity and did not provoke any inhibitory effects on P2X receptors which had not been used as selection target. The activity of the aptamers selected using recombinant receptors as targets in inhibiting wild-type P2X4 or P2X2 receptors was verified in whole-cell recording experiments with PC12 cells which endogenously express both receptor subtypes. In addition of having developed aptamers as tools to elucidate P2X2 and P2X4 receptor functions during neuronal differentiation, these nuclease-resistant aptamers are suitable for in vivo use and may turn into therapeutics in the inhibition of purinergic receptor participation in pathophysiological conditions.
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Lindner, Anna. "Untersuchung der Interaktion der Untereinheiten im humanen P2X2- und P2X2/3-Rezeptor durch Cystein-substituierte Aminosäuren." Doctoral thesis, Universitätsbibliothek Leipzig, 2015. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-189913.
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P2X-Rezeptoren treten aufgrund ihrer Präsenz in verschiedensten Organsystemen des menschlichen Körpers zunehmend in den Mittelpunkt zahlreicher Forschungsansätze. Besonderes Interesse gilt dabei u.a. den P2X2/3-Rezeptoren, da in ihnen ein neuer Angriffspunkt für die Entwicklung von Schmerztherapeutika gesehen wird. Trotz enormer Fortschritte in diesem Bereich, bleiben die Vorgänge und strukturellen Gegebenheiten, die zur Öffnung der Ionenkanäle führen, weiterhin spekulativ.
In der vorliegenden Arbeit wurden mithilfe einer Mutagenese einzelne Aminosäuren des hP2X2-Rezeptors, welche sich in geringer Entfernung zueinander zwischen zwei Untereinheiten befanden, durch Cysteine substituiert. Die Auswahl der Aminosäuren erfolgte dabei anhand eines Homologiemodells des hP2X2-Rezeptors und des Aminosäureabgleichs zwischen den hP2X2- und hP2X3-Rezeptoren. Auf diese Weise sollte deren Interaktion über eine mögliche Ausbildung von Disulfidbrücken zwischen zwei Untereinheiten untersucht werden. Die Rezeptorfunktion wurde anschließend mittels der whole-cell patch-clamp-Technik charakterisiert. Der Rezeptor reagierte bei allen untersuchten Varianten mit einem Funktionsverlust, ein spontan öffnender Kanal konnte somit nicht generiert werden.
Durch die Kombination der verschiedenen hP2X2-Rezeptor-Cysteinmutanten mit einer hP2X3-Rezeptor-Cysteindoppelmutante, konnte gezeigt werden, dass sich die verschiedenen Untereinheiten im heterotrimeren hP2X2/3-Rezeptor nicht soweit annähern, dass eine Disulfidbrücken-Bildung zwischen den Untereinheiten möglich wird. Es konnte allerdings verdeutlicht werden, dass für die Aktivierung des heterotrimeren hP2X2/3-Rezeptors zwei funktionelle Bindungsstellen zur Kanalaktivierung ausreichen.
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Helms, Nick [Verfasser], Peter [Akademischer Betreuer] Illes, Thomas [Akademischer Betreuer] Riedel, Michael [Gutachter] Schaefer, and Ralf [Gutachter] Hausmann. "Wechselwirkungen von Agonisten und kompetitiven Antagonisten mit der Ligandenbindungsstelle des schnell desensitisierenden P2X3-Rezeptors / Nick Helms ; Gutachter: Michael Schaefer, Ralf Hausmann ; Peter Illes, Thomas Riedel." Leipzig : Universitätsbibliothek Leipzig, 2016. http://d-nb.info/1240395752/34.
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Farmer, Louise Katie. "The molecular basis of antagonism at cardiovascular P2X1 and P2X4 receptors." Thesis, University of Leicester, 2014. http://hdl.handle.net/2381/40322.
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Structural information for the zebrafish P2X4 receptor in both an agonist bound and unbound resting state provided a major advance in understanding agonist action and has given insight into movement that occurs in the receptor upon ATP binding. Despite agonist action now being well characterised, the molecular basis of antagonism is poorly understood. In this thesis the mechanism of antagonist action at the hP2X1 receptor has been investigated through determining properties of chimeras and mutant receptors based on differences between antagonist sensitive and insensitive P2X receptors. The antagonists suramin, NF449 and PPADS potently inhibit the human P2X1 receptor but have little or no action at the rat P2X4 receptor. The extracellular loop of the hP2X1 receptor was shown to determine antagonist sensitivity and was therefore split into four sections, residues of which were swapped with corresponding residues of the antagonist insensitive rP2X4 receptor and vice versa. Sub-chimeras and point mutations were then made to identify particular residues and regions which contribute to antagonist action. These experiments identified two regions important for NF449 binding at the receptor. These are a cluster of four positively charged residues at the base of the cysteine rich head region (136-140) and three residues located just below them (T216, H224 and Q231). An NF449 bound model of the hP2X1 receptor has been generated. The introduction of the four positively charged residues at the base of the cysteine rich head region to the rP2X4 receptor introduced suramin and PPADS sensitivity to this previously insensitive receptor. This mutation is thought to cause a conformational change which allows the antagonist to bind at residues which are already present in the wildtype receptor. In summary this thesis has advanced the understanding of antagonist action at the hP2X1 receptor and the antagonist insensitivity of the rP2X4 receptor.
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Noack, Rebecca Verfasser], Axel [Akademischer Betreuer] Methner, and Dieter [Akademischer Betreuer] [Willbold. "Resistance against Oxidative Glutamate Toxicity: Functional Characterization of Amino Acid Antiporter Subunit xCT, Purinergic ATP Receptor P2X3 and Mitochondrial Fission Factor GDAP1 / Rebecca Noack. Gutachter: Axel Methner ; Dieter Willbold." Düsseldorf : Universitäts- und Landesbibliothek der Heinrich-Heine-Universität Düsseldorf, 2012. http://d-nb.info/1023128357/34.
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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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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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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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Lemoine, Damien. "Mise au point d'outils optogénétiques pour la photorégulation de l'activité des récepteurs canaux P2X." Thesis, Strasbourg, 2013. http://www.theses.fr/2013STRAJ072/document.
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Les récepteurs canaux P2X, sélectifs des cations, sont activés par l'ATP extracellulaire. Les récepteurs P2X remplissent de nombreux rôles physiologiques allant de la nociception à la neuromodulation. L'étude du rôle physiopathologique de ces récepteurs souffre d'un manque d'outils pharmacologiques sélectifs. L'optogénétique pharmacologique serait une méthode pour palier ce manque. Mes travaux se divisent en deux parties, l'une concernant l'étude structurale des récepteurs P2X et l'autre présentant le développement d'outils optogénétiques chimiques pour contrôler l'activité des récepteurs P2X. Dans une première série d'expériences nous avons identifié le site de liaison de l'ATP par marquage d'affinité dirigé à l'aide d'un analogue de l'ATP thiol réactif. Ensuite,nous avons démontré le mécanisme d'activation des récepteurs P2X dans une étude utilisant la bioinformatique et l'ingénierie de site zinc. Ainsi nous avons établi une corrélation entre l'ouverture du canal et le rétrécissement du site de liaison suite à la fixation de l'ATP. Enfin nous avons mis au point une nouvelle stratégie optogénétique chimique appelée « optogating » permettant de reprogrammer un canal ionique afin de le contrôler par la lumière. Nous avons montré qu'un récepteur canal modifié au niveau transmembranaire, par un réactif contenant un azobenzène, peut être activé réversiblement par la lumière sans recourir au ligand endogène. Nous avons réussi à photocontrôler l'activité neuronale à l'aide d'un récepteur P2X activé par la lumière,dans lequel, la sensibilité à l'ATP a été génétiquement supprimée. Cet outil est prometteur pour l'étude du rôle physiologique des récepteurs P2X in vivoThe ATP-gated P2X receptors are trimeric ion channels that are selective to cations.These ion channels are involved in various physiological processes such as nociception and neuromodulation. The study of P2XR physiology suffers from a lack of selective pharmacological molecules. Optogenetic pharmacology could solve this problem. ln thiswork, 1 performed structural studies of P2X receptors and developed an original optochemical tool in order to contrai P2X activity. First, we localized the ATP-binding sites by creating, through a proximity-dependent"tethering" reaction, covalent bonds between a synthesized ATP-derived thiol-reactiveP2X2 agonist (NCS-ATP) and single cysteine mutants engineered in the putativebinding cavities of the P2X2 receptor. Next, we demonstrated that tightening of the ATP-binding sites correlates precisely with channel opening in the P2X2 receptor. Finally, we developed a unique and versatile method, in which the gating machinery of the P2X2 receptor was reprogrammed to respond to light. We found that channels covalently modified by azobenzene-containing reagents at the transmembrane segments could be reversibly turned on and off by light, without the need of the natural ligand (here ATP). We demonstrated photocontrol of neuronal activity by a light-gatedP2X receptor, in which the natural sensitivity to ATP was genetically removed. These light-gated P2X receptors represent valuable tools for investigating the physiological functions of P2X receptors
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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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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 déterminerBackground : 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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Compan, Vincent. "Étude moléculaire et fonctionnelle de l'assemblage des sous-unités P2X à la membrane plasmique : caractérisation des interactions hétéromériques et oligomériques de la sous-unité P2X5 et mise en évidence de nouveaux complexes de signalisation associés au récepteur P2X2 dans les neurones." Montpellier 1, 2008. http://www.theses.fr/2008MON1T019.
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Les récepteurs ionotropiques P2X activés par l'ATP extracellulaire sont formés par l'association de trois sous-unités au sein de complexes homo- ou hétéro-mériques. In vitro, de nombreuses interactions hétéromériques ont été proposées mais peu d'entre elles ont été validées in vivo. Durant ma thèse, nous avons caractérisé in vitro et in vivo de nouveaux complexes membranaires formés par les sous-unités P2X. Une partie de ce travail s'est focalisée sur la sous-unité P2X5. Cette sous-unité forme des récepteurs homotrimériques membranaires dont la conformation particulière de la boucle extracellulaire expliquerait leur faible expression fonctionnelle. In vitro, P2X5 interagit à la surface des cellules avec les sous-unités P2X2 ou P2X4. L'association P2X5/P2X2 a été confirmée sur des tissus natifs et la stoechiométrie des hétéromères déterminée. Enfin, les homomères P2X2 et P2X5 interagissent également au sein de complexes oligomériques. Après stimulation par l'ATP, les récepteurs P2X2/P2X5 présentent une cinétique lente de perméabilité aux larges cations et induisent l'apparition de blebs à la surface des cellules. Enfin, au sein des récepteurs P2X2/P2X5, les changements conformationnels de la queue C-Term de P2X2 induits par l'ATP sont bloqués. Dans ubne dernière partie, les complexes de signalisation associés au récepteur P2X2 ont été étudiés. Dans les neurones, P2X2 est associé à VILIP1 et à la synapsine2b. L'interaction VILIP1/P2X2 est constitutive mais subit un changement conformationnel coordonnée des deux protéines lors de l'application d'ATP. Ces résultats décrivent et caractérisent de nouveaux complexes protéiques membranaires comprenant les sous-unités P2X2 et/ou P2X5.
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Peverini, Laurie. "Conception et application de nouveaux outils photochimiques pour l’étude des récepteurs canaux P2X." Thesis, Strasbourg, 2017. http://www.theses.fr/2017STRAF061/document.
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Les récepteurs P2X (P2XR), activés par l’ATP, sont impliqués dans des rôles physiopathologiques. Leur fonctionnement est associé à différents états conformationnels. Le projet de thèse a mené à associer la synthèse organique et l’application de molécules photo-activables avec des techniques d’électrophysiologie patch-clamp, pour décortiquer les mouvements moléculaires de ces récepteurs et effectuer des relations structure-fonction, via trois stratégies : - La synthèse et application d’agrafes photo-isomérisables qui permet le photo-contrôle des P2XR et l’étude de mouvements - La synthèse et caractérisation d’un acide aminé (aa) photo-clivable pour étudier les implications de zones sur la fonction des P2XR via une photolyse - L’incorporation d’un aa non naturel dans les P2XR pour étudier des interactions et mouvements via un « photo-pontage ». Nous avons élucidé les mécanismes moléculaires responsables de la perméabilité des P2XR, récusé l’existence de l'état dilaté et identifié un cation organique physiologique pouvant les traverser. Nous avons aussi conçu un acide aminé photo-clivable pouvant mener à des études structure-fonction des P2XRP2X receptors are cationic ligand-gated ion channels, activated by extracellular ATP, involved in many physio-pathological roles. Their function is associated with different allosteric states. During this PhD, we have designed three new strategies, spanning photochemical organic synthesis and patch-clamp electrophysiology to elucidate the molecular mechanisms involved in these conformational states and to collect data in order to study structure-function relationships. - Synthesis and application of molecular tweezers, which allows the photo-control of P2X Rand the study of molecular motions - Synthesis and characterization of a photo-cleavable amino acid with the aim of incorporating it into P2XR and doing structure-function relationships - Incorporation of an unnatural amino acid for photo-crosslinking studies. We have been able to probe the molecular mechanism involved in large organic cations permeation of P2XR, to bring into question the dilated state and to identify a physiological cation that can flow through P2XR. We have also designed a photo-cleavable amino acid which could serve in the study of structure-function relationships
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Bhattacharya, Sumit. "Contribution of Purinergic Receptors to Calcium Signaling in Salivary Gland." University of Toledo Health Science Campus / OhioLINK, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=mco1353370433.
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Beudez, Juline. "Etude mécanistique des récepteurs P2X par l'utilisation de molécules photoisomérisables." Thesis, Strasbourg, 2018. http://www.theses.fr/2018STRAJ084/document.
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Les récepteurs P2X, activés par l’ATP extracellulaire et cations non-sélectifs, sont impliqués dans de nombreux rôles physiopathologiques. Le manque de sélectivité de molécules pharmacologiques est un inconvénient majeur pour leur étude. La résolution de leurs structures cristallographiques a permis de les comprendre à l’échelle moléculaire, cependant les mécanismes impliqués dans les transitions allostériques restent mal compris. Au laboratoire, deux outils, dérivés d’azobenzène, permettant l’activation des récepteurs P2X en absence d’ATP et par la lumière ont été développés. L’utilisation de ces outils ont permis l’étude de la transition allostérique de l’état ouvert à l’état désensibilisé, mettant en avant une zone de régulation efficace dans les espaces transmembranaires. De plus, leur utilisation a permis l’investigation biophysique d’une mutation présente sur P2X2 humain, responsable d’une surdité non-syndromatique. Cette mutation entraine un rétrécissement du pore, impactant le passage de gros cations impliqués dans le processus d’audition. Enfin, la relation entre le diamètre du pore ionique et le passage de gros cations a été établiP2X receptors, activated by extracellular ATP and non-selective cations, are involved in many physiopathological roles. The lack of selectivity of pharmacological molecules is a major drawback for their study. The resolution of their crystallographic structures provided a molecular framework, but the mechanisms involved in allosteric transitions remain misunderstood. In the laboratory, two tools have been developed, derived from azobenzene, allowing the activation of P2X receptors in the absence of ATP and by light. The use of these tools allowed the study of the allosteric transition from the open to the desensitized state, highlighting an effective regulatory zone in transmembrane spaces. In addition, their use provided the biophysical investigation of a mutation present on hP2X2, responsible for non-syndromatic hearing loss. This mutation leads to a narrowing of the pore, affecting the large cations flow involved in hearing process. Finally, the relationship between the diameter of the ionic pore and the passage of large cations has been established
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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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Habermacher, Chloé. "Exploration structurale et dynamique du phénomène d'activation des récepteurs P2Xs par de nouveaux outils optochimiques." Thesis, Strasbourg, 2015. http://www.theses.fr/2015STRAJ050/document.
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Les récepteurs purinergiques P2Xs sont impliqués dans de nombreux processus physiopathologiques et représentent des cibles thérapeutiques majeures. Leur étude souffre néanmoins d’un manque de molécules pharmacologiques sélectives des différents sous-types et les mécanismes impliqués dans les transitions allostériques restent putatifs.Nous avons développé des outils optochimiques innovants, dérivés d’azobenzènes permettant une maîtrise spatiale et temporelle inégalée de la fonctionnalité du récepteur : d’une part,un outil dérivé d’une stratégie de pharmacologie optogénétique contrôlant l’activité d’un récepteur ingéniéré et d’autre part, une sonde moléculaire capable d’induire des mouvements entre des résidus au sein de la protéine et d’étudier les mécanismes lors de l’activation. Ces travaux nous ont permis de proposer un nouveau mécanisme d’activation du récepteur. Ces deux outils pourraient être utilisés sur d’autres cibles pour des investigations moléculaires et physiologiquesPurinergic P2X receptors are implicated in a diverse range of physiopathological processes and are therefore promising therapeuthic target. Their study suffers from the lack of pharmacological tools selective of one subtype only and mechanisms by which the receptor switches between different conformational states remain elusive.We have developed novel optochemical tools based on azobenzene derivatives to obtain a spatio temporal control of the functionality of the receptor. Inspired by optogenetic pharmacology, we have designed an engineered receptor to control electrical activity of cultured neurons. Molecular photo-switchable tweezers have been developed to explore allosteric transitions of the protein and giving new insights into the P2X pore gating mechanism. This approach provides data enabling us to purpose a new model of the active state. The versability of the two strategies makes these tools promising for molecular and physiological studies of other membrane proteins
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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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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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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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Hua, Jennifer. "Rôle des récepteurs P2X4 dans la dégradation d’ApoE : implication dans la maladie d’Alzheimer." Thesis, Montpellier, 2019. http://www.theses.fr/2019MONTT021/document.
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Les récepteurs purinergiques P2X4 (P2X4R) sont des récepteurs canaux exprimés par lesneurones et les microglies du système nerveux central et sont impliqués dans de nombreuxprocessus physiologiques et pathologiques. Des études préliminaires, menées au sein dulaboratoire, ont permis de mettre en évidence une interaction entre P2X4R etl’Apolipoprotéine E (ApoE), ainsi qu’une augmentation d’ApoE dans les macrophages et lesmicroglies provenant de souris déficientes pour P2X4R. Basée sur ces observations, lapremière partie de cette thèse a cherché à caractériser les mécanismes impliquant P2X4R danscet effet. ApoE étant un facteur de risque majeur dans la maladie d’Alzheimer, la deuxièmepartie de cette thèse a été consacrée à étudier l’implication de P2X4R dans cette pathologie.Les résultats présentés montrent que P2X4R module l’activité de la cathepsine B, enzymeresponsable de la dégradation lysosomale d’ApoE. L’utilisation de souris APP/PS1 a permisde montrer que l’absence de P2X4R conduit à une amélioration des capacités mnésiques, unediminution de la concentration de peptide Aβ soluble ainsi qu’à une augmentation d’ApoEmicrogliale.Ces résultats indiquent que P2X4R régule la dégradation d’ApoE par un mécanismedépendant de la cathepsine B, et que son invalidation permet d’améliorer les symptômescognitifs de la maladie d’AlzheimerP2X4 receptors (P2X4R) are purinergic ion channels expressed on neurons and microglia inthe central nervous system. They have been widely studied and have been implicated in manyphysiological and pathological processes. Previous studies conducted in the laboratoryrevealed an interaction between P2X4R and the Apolipoprotein E (ApoE), as well as anincrease in ApoE level in primary macrophages and microglia obtained from mice lackingP2X4R. Based on these results, this thesis aimed to decipher the mechanisms underlyingP2X4R regulation of ApoE levels. In addition, ApoE being a major risk factor forAlzheimer’s disease, part of this work investigated potential implications of P2X4R in thispathology.Results show that P2X4R modulates cathepsin B activity, which in turn promotes ApoElysosomal degradation. APP/PS1 mice lacking P2X4R show an increase in cognitiveperformances, a decrease in soluble Aβ peptide and an increase of microglia ApoE level.These results support that P2X4R modulates ApoE degradation in a cathepsin B-dependantmanner and that its invalidation leads to an improvement in Alzheimer’s pathology
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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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Qureshi, Omar Saleem. "Targeting and trafficking of P2X4 receptors." Thesis, University of Cambridge, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.613833.
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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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Gu, Baijun. "THE P2X7 RECEPTOR OF HUMAN LEUKOCYTES." University of Sydney. Medicine, 2003. http://hdl.handle.net/2123/501.
Full textAbstract:
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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Rayah, Amel. "Identification des voies biochimiques stimulées par le récepteur purinergique P2X7 qui sont impliquées dans le clivage protéolytique du précurseur de la protéine amyloïde (APP)." Thesis, Paris 11, 2015. http://www.theses.fr/2015PA11T043.
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Le précurseur de la protéine amyloïde (APP) est une protéine transmembranaire qui, après coupure séquentielle par les sécrétases β et γ, produit des peptides Aβ trouvés dans les plaques séniles de patientsatteints d’Alzheimer. Par contre, la forme soluble de l’APP (sAPPα), produite après coupure par une sécrétase α, augmente la survie cellulaire, la croissance des neurites et la synaptogénèse. L’APP est coupéeau site α par 3 métalloprotéases : ADAM9, ADAM10 et ADAM17.Notre laboratoire a montré que la stimulation du récepteur purinergique P2X7 (P2X7R) provoque la coupure protéolytique du précurseur de la protéine amyloïde (APP). Le Dr Delarasse a établi que la voie non amyloïdogénique est mise en jeu et que c'est le fragment sAPPα, neuroprotecteur, qui est produit. Deplus, le laboratoire a précédemment démontré que ce ne sont pas les alpha-sécrétases ADAM9, 10 et 17 qui sont responsables du clivage protéolytique de l'APP après stimulation du P2X7R dans les cellules de neuroblastome Neuro2a.Durant mes travaux de thèse, nous avons étudié la voie biochimique menant à la libération du fragments APPα. L’activation du P2X7R stimule la phosphorylation et la translocation rapide à la membrane plasmique de protéines, appelées ezrine, radixine et moesine (ERM) qui ont la capacité d’établir un lien entre la région cytosolique du P2X7R et la F-actine. Les ERM jouent un rôle crucial dans la coupure protéolytique de l’APP par les métalloprotéases ADAM. En effet, l’inhibition de l’expression des ERM par RNA interférence aboutit à une absence de coupure de l’APP. Par ailleurs, nous avons observé que les MAPKERK1/2 et JNK et la ROCKinase sont nécessaires à la phosphorylation activatrice des ERM et jouent donc un rôle en amont des ERM. Enfin, nous avons mis en évidence le rôle de la PI3K en aval des ERM.Par ailleurs, nous avons démontré que l’activation du récepteur purinergique P2X7 entraînait la coupure protéolytique de la molécule NrCAM par ADAM17 aboutissant à la libération du fragment soluble del’ectodomaine de NrCAM. Les résultats obtenus indiquent que la coupure de NrCAM est dépendante de l’activation et de la fixation des ERM à NrCAM. Ces résultats suggèrent fortement que les ERM sont indispensables à la coupure protéolytique de différents substrats après stimulation du P2X7R.Les données obtenues mettent en évidence un mécanisme moléculaire original et important qui fait jouer aux ERM un rôle central de « liens moléculaires » dans le clivage protéolytique des protéines transmembranaires. A ce stade de notre étude, nous émettons l’hypothèse que les ERM agissent en aval du récepteur P2X7, en liant les substrats et/ou les protéases qu’ils regroupent à la membrane plasmique favorisant ainsi le clivage des substratsThe amyloid protein precursor (APP) can be cleaved in neural cells by α-secretases to produce the soluble APP ectodomain (sAPPα), which is neuroprotective. We have shown previously that activation of the purinergic receptor P2X7 (P2X7R), a member of the P2X receptor family of ATP-gated cation channels, triggers sAPPα shedding from neural cells. Here, we demonstrate that theactivation of Ezrin/Radixin/Moesin proteins (ERM) is required for the P2X7R-dependent proteolyticprocessing of APP leading to sAPPα release. Indeed, the down regulation of ERM by siRNA blocksthe P2X7R-dependent shedding of sAPPα. We also show that P2X7R stimulation triggers thephosphorylation of ERM. Thus, ezrin translocates to the plasma membrane to interact with P2X7R.Using specific pharmacological inhibitors, we have established the order in which several enzymestrigger the P2X7R-dependent release of sAPPα. Thus, a Rho-kinase and the MAPK modules ERK1/2and JNK act upstream of ERM while a PI3Kinase activity is triggered downstream. This work for the first time identifies ERM as major partners in the regulated non-amyloidogenic processing of APP. Inaddition, we have recently established that the stimulation of P2X7R leads to the proteolytic cleavage of NrCAM by ADAM17 and the shedding of the soluble extracellular domain of NrCAM. Our results clearly show that the proteolytic cleavage of NrCAM is dependant of ERM activation and fixation tothe intracellular region of NrCAM. Thus, our results strongly suggest that ERM are required for the proteolytic cleavage of numerous substrates after P2X7R stimulation. Our findings suggest that ERM play a central role in the proteolytic cleavage of transmembrane proteins and act as molecular linkswhich aggregate ADAMs and substrates at the plasma membrane promoting the cleavage of substrates
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Jones, Clare Alexa. "Molecular pharmacology of P2X{sub4} and P2X{sub6} receptors for ATP." Thesis, University of Cambridge, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.620195.
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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 calciumExtracellular 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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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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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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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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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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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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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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