Les maladies inflammatoires intestinales (MII) sont caractérisées par des périodes d'inflammation chronique entrecoupées de périodes de rémission. Elles mobilisent l'action de plusieurs molécules pour favoriser le retour à l'homéostasie. Il a été démontré que l'expression du récepteur P2Y[indice inférieur 2] (P2Y[indice inférieur 2]) est augmentée chez les patients atteints de MII et dans un modèle murin de colite chimique. Cette thèse s'intéresse aux mécanismes de régulation transcriptionnelle du gène de P2Y[indice inférieur 2] et à son rôle dans les MII. Premièrement, la disponibilité de la chromatine au promoteur de P2Y[indice inférieur 2] a été caractérisée par le statut d'acétylation des histones H3 et H4 par immunoprécipitation de la chromatine (ChIP). Le site d'initiation de la transcription du gène de P2Y[indice inférieur 2] a été identifié par 5' RLM-RACE. NF-[kappa]B et C/EBP[bêta] sont des candidats idéals pour réguler l'expression de P2Y[indice inférieur 2] puisqu'ils sont impliqués dans la régulation de gènes liés à l'inflammation. En utilisant le logiciel TRANSFAC, les sites de liaison potentielle (SLP) de NF-[kappa]B et C/EBP[bêta] ont été identifiés sur le promoteur du récepteur P2Y[indice inférieur 2]. Ensuite, des essais luciférase ont démontré que NF-[kappa]B et C/EBP[bêta] transactivent le promoteur de P2Y[indice inférieur 2] dans les cellules épithéliales intestinales (CEI) en condition inflammatoire ou non. Lorsque ces deux facteurs de transcription sont co-exprimés dans les CEI, on observe une synergie de la transactivation du promoteur de P2Y[indice inférieur 2]. Par des essais luciférase avec des constructions du promoteur mutées pour les SLP des facteurs de transcription à l'étude ainsi que par des gels de retardement et des essais de ChIP, nous avons montré que C/EBP[bêta] se lie en position -229 à -220pb du promoteur et que NF-[kappa]B se lie en position -181 à -172pb. Finalement, la stimulation du récepteur P2Y[indice inférieur 2] active la voie de signalisation PI-3K/Akt qui inactive GSK-3[bêta] favorisant la stabilisation des microtubules permettant à la cellule de migrer pour réparer une monocouche de CEI blessées. De plus, dans les CEI, l'expression de COX-2 et PGE[indice inférieur 2] qui sont des molécules importantes pour la réparation tissulaire, est augmentée par la stimulation de P2Y[indice inférieur 2]. In vivo, la stimulation du récepteur favorise la rémission de souris atteintes de colite chimique. Cette thèse démontre donc que l'expression du récepteur P2Y[indice inférieur 2] dans les MII est régulée par NF-[kappa]B et C/EBP[bêta] et que ce récepteur joue un rôle important dans les MII en favorisant la phase de rémission.

In light of the significant modulation of receptor activity previously shown by a peptide (designated L247), designed to mimic the third extracellular loop of the human P2Y2 receptor, the aim of this study was to use this peptide as an immunogen to generate and fully characterise polyclonal rabbit antibodies to the P2Y2 receptor. Other aims of this study were to characterise epithelial and endothelial cells for a thorough expression profile of P2Y receptor mRNA transcripts in order to provide a rapid screen for the molecular determinants of these receptors in these cells. These studies also aimed to confirm previously published pharmacology, thus, to set the basis for western blot studies using P2Y receptor antibodies. Bovine aortic endothelial cells that co-express P2Y1 and P2Y2 receptors; EAhy926, a human endothelial fusion cell line, that express P2Y2 receptors; and ECV304 human bladder cancer cell line, known to express P2Y2-like and P2Y11-like receptors were used in this study. The dose dependent accumulation of inositol phosphates and cAMP response to potent P2Y11 agonists and RT-PCR studies confirmed the functional expression of both P2Y2 and P2Y11 receptors in ECV304 cells. Likewise, the dose dependent accumulation of inositol phosphates in response to potent P2Y2 and P2Y6 agonists and the presence of mRNA transcripts confirmed the expression of functional P2Y2/4- like and P2Y6- like receptors in EAhy926 cells. Polyclonal antiserum raised against L247 peptide was affinity purified and the purified fractions showed strong immunoreactivity with immobilised immunogenic antigen in ELISA. In western blot analysis L247 rabbit polyclonal anti-P2Y2 antibody detected strong bands in ECV304 and EAhy926 cells. On pre-absorption with the immunogenic peptide these responses were abolished suggesting that this antibody is antigen specific. Agonist induced P2Y2 receptor desentisation studies in ECV304 cells showed that prolonged agonist incubation caused the receptor sequestration. The loss of bands caused by P2Y2 receptor desensitisation and sequestration in membrane enriched fractions of agonist incubated ECV304 cells confirmed the specificity of L247 antibody. This antibody also showed no immunoreactivity in 1321N1 human brain astrocytoma cells devoid of any P2Y receptor subtypes cells. Deglycosylation studies revealed that the P2Y2 receptors are glycosylated in ECV304 cells. The polyclonal rabbit anti-P2Y2 receptor antibodies obtained from commercial sources produced completely different immunoreactive profiles with multiple bands even in 1321N1 cells. Furthermore, in comparison to L247 anti-P2Y2 antibody the commercial antibody showed no difference between normal and agonist incubated cells suggesting that this antibody may not be recognising the P2Y2 receptors in ECV304 cells. Likewise polyclonal rabbit antibodies to other P2Y receptors either showed no response or showed strong immunoreactive profile with multiple bands even in 1321N1 cells suggesting that these antibodies may not have been extensively characterized. Furthermore, immunofluorescence studies with commercial anti-P2Y2 antibodies showed that they may be only recognising non-denatured receptors. These studies suggest that the L247 anti-P2Y2 antibody raised against peptide designed to mimic specific region in the third extracellular loop of human P2Y2 receptor is highly specific and sensitive and provides an important tool to study endogenously expressed P2Y2 receptors in both non-denatured and denatured state. These studies indicate that this strategy of generating antibodies may be used to generate highly specific antibodies to other P2Y receptor subtypes.

Les récepteurs P2Y sont exprimés à la surface de bon nombre de cellules y compris les cellules du système immunitaire. De nombreuses études réalisées in vitro suggèrent une influence des nucléotides sur la biologie de ces cellules. Toutefois pour certains d’entre eux, l’implication in vivo n’est pas clairement établie. Le présent travail, basé sur l’analyse phénotypique de souris invalidées, vise donc à étudier le rôle de trois de ces récepteurs, à savoir P2Y2, P2Y6 et P2Y12, dans la physiologie des phénomènes inflammatoires. Leur rôle a été abordé plus particulièrement dans deux types cellulaires :les macrophages et les cellules dendritiques.<p>En ce qui concerne les macrophages péritonéaux recrutés, nous avons démontré in vitro que le récepteur P2Y2 participe, avec un récepteur P1, à la production d’IL-6 en réponse au LPS. Par contre, il ne semble pas impliqué dans l’internalisation d’antigènes par macropinocytose. Le récepteur P2Y6 favorise la production d’IL-6 et de MIP-2 en réponse au LPS et stimule l’endocytose par macropinocytose de dextran-FITC.<p>Pour ce qui est des cellules dendritiques (DCs), l’étude du rôle du récepteur P2Y12 dans leur biologie in vitro avait été investigué auparavant dans notre laboratoire. Toutefois, certains paramètres devaient être confirmés ainsi que l’implication in vivo du récepteur dans l’établissement d’une réponse immune. Nous avons contribué à démontrer que l’endocytose par macropinocytose est augmentée suite à l’activation du récepteur P2Y12 par l’ADPβS au niveau de DCs spléniques murines et de DCs dérivées de monocytes humains. L’ADPβS favorise la capacité des DCs à activer les lymphocytes T spécifiques d’un antigène. Enfin, la signification de ces observations a été testé in vivo par l’immunisation de souris P2Y12+/+ et P2Y12-/-. Les résultats obtenus montrent une production d’IgG1 diminuée dans les souris invalidées pour le récepteur par rapport aux souris sauvages suggérant que le récepteur P2Y12 favoriserait le développement d’une réponse immune Th2 et ce principalement via l’augmentation de la captation d’antigènes par les DCs. <p>Suite aux données obtenues et à celles de la littérature, nous avons investigué l’implication du récepteur P2Y6 dans la réponse immune adaptative in vivo. Pour ce faire, nous avons immunisé des souris P2Y6+/+ et P2Y6-/- selon le protocole utilisé lors de notre précédente étude. Les souris P2Y6 KO montrent une diminution significative de la production d’IgG2c spécifiques de l’antigène suggérant l’implication du récepteur dans le développement d’une réponse pro-Th1. Plusieurs paramètres ont été mesurés pour expliquer ce phénomène. Nous avons tout d’abord démontré que le récepteur P2Y6 est exprimé et fonctionnel dans les cellules dendritiques dérivées de la moelle. Nous avons ensuite montré que l’activation du récepteur par l’UDP favorise la capture d’antigènes in vitro, potentialise l’expression de molécules de co-stimulation (CD80 et CD86) et la production d’IL-12 p70 et de TNF-α en réponse au CpG ODN indiquant que la modulation de la réponse immune observée in vivo s’explique, en partie du moins, par les effets du récepteur sur différentes fonctions des DCs.<p> / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Magíster en fisiología / La hipertensión arterial pulmonar (HAP) es una enfermedad crónica, progresiva y fatal. A pesar del desarrollo de nuevas drogas, continúa siendo incurable. Las tres vías de tratamiento farmacológico actual apuntan a la regulación del tono vascular pulmonar, sin embargo, no se ha considerado la posible participación del sistema de regulación purinérgica del tono, el cual ya es conocido por su contribución a la regulación de la vaso-actividad a nivel sistémico. La escasa bibliografía disponible que ha permitido iniciar la caracterización del sistema purinérgico a nivel pulmonar se refiere solo al nivel arterial, sin embargo, los vasos venosos intrapulmonares pequeños dan cuenta de cerca de un 20% de la resistencia vascular pulmonar, lo que hace de este un componente importante en HAP. Es por esto que nuestro objetivo fue evaluar la contracción de venas intrapulmonares pequeñas inducida por UDP y buscar el receptor sensible a UDP involucrado (P2Y6 o P2Y14 ). Registramos la contracción de venas intrapulmonares pequeñas (VIP) inducida por dosis crecientes de UDP en rebanadas de pulmón de ratas con HAP y ratas sanas; luego desarrollamos ensayos funcionales con agonistas y antagonistas específicos para los receptores P2Y6 y P2Y14 y finalmente evaluamos la expresión de dichos receptores a través de Inmunofluorescencia Indirecta (IFI). Nuestros resultados muestran por primera vez que el UDP es un agonista capaz de inducir contracción dependiente de concentración en VIP tanto en ratas sanas como HAP, en este último grupo se observa una contracción mediada por UDP de una magnitud mayor a la lograda en ratas sanas. Los estudios funcionales con agonistas específicos para cada receptor muestran contracción mediada por el receptor P2Y6 y en muy menor medida por el receptor P2Y14 en ratas sanas e interesantemente demostramos una hipercontractilidad mediada por UDP-Glucosa a través de su único receptor P2Y14 en ratas HAP. La IFI ratifica la presencia del receptor P2Y6 en músculo liso de ratas controles e hipertensas y consecuentemente con los estudios funcionales se demostró una mayor presencia del receptor P2Y14 en músculo liso de VIP de ratas con HAP en comparación a ratas sanas. Nosotros concluimos que es el receptor P2Y14 el que presenta una destacada participación en la fisiopatología de la HAP; esto debido a que su activación específica induce una respuesta hipercontráctil de las VIP en ratas enfermas, lo cual se ve respaldado por su mayor colocalización a nivel del músculo liso en la pared de VIP de ratas HAP. Esto nos permite pensar en el receptor P2Y14 como un nuevo target de acción farmacológica para el tratamiento de la enfermedad.

Die hohe Dichte und breite Verteilung von P2Y-Rezeptoren im Hirn von Säugetieren lässt für diese Rezeptoren eine wichtige Rolle in den Prozessen des zentralen Nervensystems vermuten. Um die Beteiligung von P2Y-Rezeptoren bei der Verarbeitung von Angst zu untersuchen, wurde in dieser Arbeit das P2Y1,11,12,13 -rezeptorspezifische ADP-Analogon Adenosin-5\''-O-2-thiodiphosphat (ADPßS), der P2X1,3-Rezeptoragonist a,b-methylen-ATP (a,bmeATP), der unspezifische P2-Rezeptorantagonist Pyridoxalphosphat-6-azophenyl-2’,4’-disulfonat (PPADS) und der spezifische P2Y1-Rezeptorantagonist N6-methyl-2’-deoxyadenosin-3’:5’bisphosphat (MRS 2179) Ratten intracerebroventrikulär injiziert und die Wirkung in einem Verhaltensversuch im standardisierten Angstmodell des elevated plus-maze untersucht. Die Substanzen wurden zu 0,5 μl verabreicht. ADPßS (50 fmol und 500 fmol) zeigte dabei anxiolytische Wirkung mit vermehrten Eintritten und gesteigerter Aufenthaltszeit der Tiere auf den offenen Armen. Eine Prämedikation mit PPADS (5 pmol) oder MRS 2179 (5 pmol) konnte diesen Effekt vollständig antagonisieren. Auch eine Vorbehandlung durch den unspezifischen NO-Synthase-Inhibitor Nw-nitro-L-arginin-methyl-ester (L-NAME) konnte die ADPβS-Wirkung verhindern. Bei alleiniger Gabe zeigten diese drei Substanzen anxiogene Wirkung mit einer verminderten Aufenthaltszeit und einer geringeren Zahl von Eintritten in die offenen Armen. Der anxiogene Effekt konnte wiederum durch eine Gabe von L-Arginin (500 pmol), einem Substrat der NO-Synthase (NOS), verhindert werden, nicht aber durch das Enantiomer D-Arginin (500 pmol), das kein Substrat der NOS darstellt. Die doppelte Immunfluoreszenz konnte die Präsenz der P2Y1-Rezeptoren an Neuronen in dorsomedialen Hypothalamus, Amygdala, Hippokampus und zentralen Höhlengrau wie auch die Kolokalisation von P2Y1-Rezeptoren und nNOS nachweisen. Die höchste Dichte an Immunoreaktivität fand sich im dorsomedialen Hypothalamus. Durch die lokale bilaterale Mikroinjektion von ADPßS und MRS 2179 konnten die in den vorausgegangen Versuchen erreichten Ergebnisse reproduziert und bestätigt werden. Zusammenfassend lässt sich postulieren, dass P2Y1-Rezeptoren maßgeblich an der Verarbeitung von Angst bei männlichen Wistar-Ratten beteiligt sind, die Wirkung eng mit der Veränderung der NO-Konzentration verbunden ist und dass diese im dorsomedialen Hypothalamus vermittelt wird. Inwieweit diese Mechanismen auch in Amygdala und Hippokampus eine Rolle spielen kann mit den vorliegenden Daten nicht abschließend beantwortet werden.

<p>The tubuloglomerular feedback mechanism is an important regulator in the juxtaglomerular apparatus and it detects flow dependent alterations in luminal NaCl concentration ([NaCl]) at the macula densa (MD) cell site via a Na+-K+-2Cl cotransporter. Signals are sent by the MD to adjust the afferent arteriole tone and altering release of renin. This signaling mechanism is unclear but MD cell calcium concentration, release of ATP and nitric oxide (NO) might be important.</p><p>In cultured rat glomerular mesangial cells the NO production was measured using confocal microscopy and calcium responses to ATP was measured with fura-2 using imaging techniques. NO from spermine-NONOate and L-arginine could resensitize, desensitized ATP receptors in a cGMP independent way. In mesangial cells from spontaneously hypertensive rats (SHR) less NO effect was found on ATP receptor de/resensitization indicating an impaired NO release or effect.</p><p>The macula densa cells were studied using microperfusion techniques with confocal and video imaging systems. Changes in [Ca2+]i from exposed macula densa plaques were assessed upon addition of agonists added to bath. The order of efficacy of agonists was UTP = ATP >> 2MesATP = ADP. Dose response curve for ATP added in bath showed an EC50 of 15 μM. Macula densa cell volume and NO concentration increased considerably with increasing luminal [NaCl] indicating an important role for NO in the signaling process to counteract a vasoconstrictor response and reset the sensitivity of the tubuloglomerular feedback mechanism. </p><p>In conclusion, the results showed 1). NO can increase the P2Y receptor resensitization in rat glomerular mesangial cells, acting through a cGMP-independent pathway. 2) An impaired NO generation/effect on P2Y receptors in mesangial cells from SHR rats. 3) Macula densa cells possess P2Y2, purinergic receptors on basolateral and that activation of these receptors results in the mobilization of Ca2+. 4) Increased luniinal [NaCl] delivery increased cell volume and the NO productions in the macula densa cells. </p>

The tubuloglomerular feedback mechanism is an important regulator in the juxtaglomerular apparatus and it detects flow dependent alterations in luminal NaCl concentration ([NaCl]) at the macula densa (MD) cell site via a Na+-K+-2Cl cotransporter. Signals are sent by the MD to adjust the afferent arteriole tone and altering release of renin. This signaling mechanism is unclear but MD cell calcium concentration, release of ATP and nitric oxide (NO) might be important. In cultured rat glomerular mesangial cells the NO production was measured using confocal microscopy and calcium responses to ATP was measured with fura-2 using imaging techniques. NO from spermine-NONOate and L-arginine could resensitize, desensitized ATP receptors in a cGMP independent way. In mesangial cells from spontaneously hypertensive rats (SHR) less NO effect was found on ATP receptor de/resensitization indicating an impaired NO release or effect. The macula densa cells were studied using microperfusion techniques with confocal and video imaging systems. Changes in [Ca2+]i from exposed macula densa plaques were assessed upon addition of agonists added to bath. The order of efficacy of agonists was UTP = ATP &gt;&gt; 2MesATP = ADP. Dose response curve for ATP added in bath showed an EC50 of 15 μM. Macula densa cell volume and NO concentration increased considerably with increasing luminal [NaCl] indicating an important role for NO in the signaling process to counteract a vasoconstrictor response and reset the sensitivity of the tubuloglomerular feedback mechanism. In conclusion, the results showed 1). NO can increase the P2Y receptor resensitization in rat glomerular mesangial cells, acting through a cGMP-independent pathway. 2) An impaired NO generation/effect on P2Y receptors in mesangial cells from SHR rats. 3) Macula densa cells possess P2Y2, purinergic receptors on basolateral and that activation of these receptors results in the mobilization of Ca2+. 4) Increased luniinal [NaCl] delivery increased cell volume and the NO productions in the macula densa cells.

Certain membrane lipids are involved in intracellular signalling processes, among them phosphoinositides and diacylglycerol (DAG). They mediate a variety of functions, including the effects of nutrients and neurohormonal stimuli on insulin secretion from pancreatic β-cells. To ensure specificity of the signal, their concentrations are maintained under tight spatial and temporal control. Here, live-cell imaging techniques were employed to investigate spatio-temporal aspects of lipid signalling in the plasma membrane of insulin-secreting β-cells. The concentration of phosphatidylinositol 4-phosphate [PtdIns(4)P] increased after stimulation with glucose or Gq protein-coupled receptor agonists. The glucose effect was Ca2+-dependent, whereas the receptor response was mediated by isoforms of novel protein kinase C (PKC). The increases in PtdIns(4)P were paralleled by lowerings of the phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] concentration. This relationship was not caused by conversion of PtdIns(4,5)P2 to PtdIns(4)P but rather reflected independent regulation of the two lipids. Stimulation of β-cells with glucose or a high K+ concentration induced pronounced, repetitive increases in plasma-membrane DAG concentration, which were locally restricted and lasted only for a few seconds. This pattern was caused by exocytotic release of ATP, which feedback-activates purinergic P2Y1-receptors and stimulates local phospholipase C-mediated DAG generation. Despite their short durations the DAG spikes triggered local activation of PKC. Novel PKCs were recruited to the plasma membrane both after glucose and muscarinic receptor stimulation. While the glucose-induced translocation was synchronized with DAG spiking, muscarinic stimulation induced sustained elevation of the DAG concentration and stable membrane association of the kinase. Also conventional PKCs translocated to the membrane after glucose and receptor stimulation. The glucose-induced response was complex with sustained membrane association mirroring the cytoplasmic Ca2+ concentration, and superimposed brief recurring translocations caused by DAG. Interruption of the purinergic feedback loop underlying DAG spiking suppressed insulin secretion. Since the DAG spikes reflected exocytosis events, a single-cell secretion assay was established, which allowed continuous recording of secretion dynamics from many cells in parallel over extended periods of time. With this approach it was possible to demonstrate that insulin exerts negative feedback on its own release via a phosphatidylinositol 3,4,5-trisphosphate-dependent mechanism.

Submitted by Alessandra Portugal (alessandradf@ioc.fiocruz.br) on 2013-09-20T15:33:03Z No. of bitstreams: 1 Anael Viana Pinto Alberto.pdf: 4150812 bytes, checksum: 9ce0a5d780533302dcc603ae65f510fe (MD5) / Made available in DSpace on 2013-09-20T15:33:03Z (GMT). No. of bitstreams: 1 Anael Viana Pinto Alberto.pdf: 4150812 bytes, checksum: 9ce0a5d780533302dcc603ae65f510fe (MD5) Previous issue date: 2012-10-31 / 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.