21 |
Caracterização dos receptores P2 em eosinófilos de ratos e do poro associado ao receptor P2X7Alberto, Anael Viana Pinto January 2012 (has links)
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.
|
22 |
Modulation of Nicotinic ACh-, GABA(a)- and 5-HT<sub>3</sub>-Receptor Functions by External H-7, a Protein Kinase Inhibitor, in Rat Sensory NeuronesHu, Hong Zhen, Li, Zhi Wang 01 December 1997 (has links)
1. The effects of external H-7, a potent protein kinase inhibitor, on the responses mediated by γ-aminobutyric acid A type (GAGA(A))-, nicotinic acetylcholine (nicotinic ACh)-, ionotropic 5-hydroxytryptamine (5-HT3)-, adenosine 5'-triphosphate (ATP)-, N-methyl-D-aspartate (NMDA)- and kainate (KA)-receptors were studied in freshly dissociated rat dorsal root ganglion neurone by use of whole cell patch-clamp technique. 2. External H-7 (1-1000 μM) produced a reversible, dose-dependent inhibition of whole cell currents activated by GABA, ACh and 5-HT. 3. Whole-cell currents evoked by ATP, 2-methylthio-ATP, NMDA and KA were sensitive to external H-7. 4. External H-7 shifted the dose-response curve of GABA-activated currents downward without changing the EC50 significantly (from 15.0 ± 4.0 μM to 18.0 ± 5.0 μM). The maximum response to GABA was depressed by 34.0 ± 5.3%. This inhibitory action of H-7 was voltage-independent. 5. Intracellular application of H-7 (20 μM), cyclic AMP (1 mM) and BAPTA (10 mM) could not reverse the H-7 inhibition of GABA-activated currents. 6. The results suggest that external H-7 selectively and allosterically modulates the functions of GABA(A)-, nicotine ACh- and 5-HT3 receptors via a common conserved site in the external domain of these receptors.
|
23 |
Regulation of Microglial Functions by Purinergic Mechanisms in the Healthy and Diseased CNSIlles, Peter, Rubini, Patrizia, Ulrich, Henning, Zhao, Yafei, Tang, Yong 17 April 2023 (has links)
Microglial cells, the resident macrophages of the central nervous system (CNS), exist in a process-bearing, ramified/surveying phenotype under resting conditions. Upon activation by cell-damaging factors, they get transformed into an amoeboid phenotype releasing various cell products including pro-inflammatory cytokines, chemokines, proteases, reactive oxygen/nitrogen species, and the excytotoxic ATP and glutamate. In addition, they engulf pathogenic bacteria or cell debris and phagocytose them. However, already resting/surveying microglia have a number of important physiological functions in the CNS; for example, they shield small disruptions of the blood–brain barrier by their processes, dynamically interact with synaptic structures, and clear surplus synapses during development. In neurodegenerative illnesses, they aggravate the original disease by a microglia-based compulsory neuroinflammatory reaction. Therefore, the blockade of this reaction improves the outcome of Alzheimer’s Disease, Parkinson’s Disease, multiple sclerosis, amyotrophic lateral sclerosis, etc. The function of microglia is regulated by a whole array of purinergic receptors classified as P2Y12, P2Y6, P2Y4, P2X4, P2X7, A2A, and A3, as targets of endogenous ATP, ADP, or adenosine. ATP is sequentially degraded by the ecto-nucleotidases and 5′-nucleotidase enzymes to the almost inactive inosine as an end product. The appropriate selective agonists/antagonists for purinergic receptors as well as the respective enzyme inhibitors may profoundly interfere with microglial functions and reconstitute the homeostasis of the CNS disturbed by neuroinflammation.
|
24 |
The Physiological Role of Serotonergic Transmission in Adult Rat Taste BudsJaber, Fadi Luc 21 May 2013 (has links)
No description available.
|
Page generated in 0.0163 seconds