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ATP-induced apoptosis involves a Ca(2+)-independent phospholipase A(2) and 5-lipoxygenase in macrophages

COSTA-JUNIOR, Helio Miranda; MENDES, Anderson Nogueira; DAVIS, Gustavo Henrique Nolasco Grimmer; CRUZA, Cristiane Monteiro da; VENTURA, Ana Lucia Marques; SEREZANI, Carlos Henrique; FACCIOLI, Lucia Helena; NOMIZO, Auro; FREIRE-DE-LIMA, Celio G.; BISAGGIO,
Fonte: ELSEVIER SCIENCE INC Publicador: ELSEVIER SCIENCE INC
Tipo: Artigo de Revista Científica
Português
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Macrophages express P2X(7) and other nucleotide (P2) receptors, and display the phenomena of extracellular ATP (ATP(e))-induced P2X(7)-dependent membrane permeabilization and cell death by apoptosis and necrosis. P2X7 receptors also cooperate with toll-like receptors (TLRs) to induce inflammasome activation and IL-1 beta secretion. We investigated signaling pathways involved in the induction of cell death by ATP, in intraperitoneal murine macrophages. Apoptosis (hypodiploid nuclei) and necrosis (LDH release) were detected 6 h after an induction period of 20 min in the presence of ATP Apoptosis was blocked by caspase 3 and caspase 9 inhibitors and by cyclosporin A. The MAPK inhibitors PD-98059, SB-203580 and SB-202190 provoked no significant effect oil apoptosis, but SB-203580 blocked LDH release. Neither apoptosis nor necrosis was inhibited when both intra- and extracellular Ca(2+) were chelated during the induction period. Mepacrine, a generic PLA(2) inhibitor and BEL, an inhibitor of Ca(2+)-independent PLA(2) (iPLA(2)) blocked apoptosis, while pBPB and AACOOPF(3). inhibitors of secretory and Ca(2+)-dependent PLA(2) respectively, had no significant effect. Cycloxygenase inhibitors had no effect on apoptosis, while the inhibitors of lipoxygenase (LOX) and leukotriene biosynthesis nordihydroguaiaretic acid (NDGA)...

Mouse Leydig cells express multiple P2X receptor subunits

ANTONIO, Ligia Subitoni; COSTA, Roberta Ribeiro; GOMES, Marcelo Damario; VARANDA, Wamberto Antonio
Fonte: SPRINGER Publicador: SPRINGER
Tipo: Artigo de Revista Científica
Português
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ATP acts on cellular membranes by interacting with P2X (ionotropic) and P2Y (metabotropic) receptors. Seven homomeric P2X receptors (P2X(1)-P2X(7)) and seven heteromeric receptors (P2X(1/2), P2X(1/4), P2X(1/5), P2X(2/3), P2X(2/6), P2X(4/6), P2X(4/7)) have been described. ATP treatment of Leydig cells leads to an increase in [Ca(2+)](i) and testosterone secretion, supporting the hypothesis that Ca(2+) signaling through purinergic receptors contributes to the process of testosterone secretion in these cells. Mouse Leydig cells have P2X receptors with a pharmacological and biophysical profile resembling P2X(2). In this work, we describe the presence of several P2X receptor subunits in mouse Leydig cells. Western blot experiments showed the presence of P2X(2), P2X(4), P2X(6), and P2X(7) subunits. These results were confirmed by immunofluorescence. Functional results support the hypothesis that heteromeric receptors are present in these cells since 0.5 mu M ivermectin induced an increase (131.2 +/- 5.9%) and 3 mu M ivermectin a decrease (64.2 +/- 4.8%) in the whole-cell currents evoked by ATP. These results indicate the presence of functional P2X(4) subunits. P2X(7) receptors were also present, but they were non-functional under the present conditions because dye uptake experiments with Lucifer yellow and ethidium bromide were negative. We conclude that a heteromeric channel...

P2X(7) receptor activation amplifies lipopolysaccharide-induced vascular hyporeactivity via interleukin-1 beta release

CHIAO, Chin-Wei; TOSTES, Rita C.; WEBB, R. Clinton
Fonte: AMER SOC PHARMACOLOGY EXPERIMENTAL THERAPEUTICS Publicador: AMER SOC PHARMACOLOGY EXPERIMENTAL THERAPEUTICS
Tipo: Artigo de Revista Científica
Português
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Lipopolysaccharide (LPS) stimulates cytoplasmic accumulation of pro-interleukin (IL)-1 beta. Activation of P2X(7) receptors stimulates conversion of pro-IL-1 beta into mature IL-1 beta, which is then secreted. Because both LPS (in vivo) and IL-1 beta (in vitro) decrease vascular reactivity to contractile agents, we hypothesized the following: 1) P2X(7) receptor activation contributes to LPS-induced vascular hyporeactivity, and 2) IL-1 beta mediates this change. Thoracic aortas were obtained from 12-week-old male C57BL/6 mice. The aortic rings were incubated for 24 h in Dulbecco`s modified Eagle`s medium, LPS, benzoylbenzoyl-ATP (BzATP; P2X(7) receptor agonist), LPS plus BzATP, oxidized ATP (oATP; P2X(7) receptor antagonist), or oATP plus LPS plus BzATP. After the treatment, the rings were either mounted in a myograph for evaluation of contractile activity or homogenized for IL-1 beta and inducible nitric-oxide synthase (iNOS) protein measurement. In endothelium-intact aortic rings, phenylephrine (PE)-induced contractions were not altered by incubation with LPS or BzATP, but they significantly decreased in aortic rings incubated with LPS plus BzATP. Treatment with oATP or IL-1ra (IL-1 beta receptor antagonist) reversed LPS plus BzATP-induced hyporeactivity to PE. In the presence of N(G)-nitro-L-arginine methyl ester or N-([3-(aminomethyl) phenyl] methyl) ethanimidamide (selective iNOS inhibitor)...

P2X4 receptors interact with both P2X2 and P2X7 receptors in the form of homotrimers

ANTONIO, L. S.; STEWART, A. P.; XU, X. J.; VARANDA, W. A.; MURRELL-LAGNADO, R. D.; EDWARDSON, J. M.
Fonte: WILEY-BLACKWELL Publicador: WILEY-BLACKWELL
Tipo: Artigo de Revista Científica
Português
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BACKGROUND AND PURPOSE The P2X receptor family consists of seven subunit types - P2X1-P2X7. All but P2X6 are able to assemble as homotrimers. In addition, various subunit permutations have been reported to form heterotrimers. Evidence for heterotrimer formation includes co-localization, co-immunoprecipitation and the generation of receptors with novel functional properties; however, direct structural evidence for heteromer formation, such as chemical cross-linking and single-molecule imaging, is available in only a few cases. Here we examined the nature of the interaction between two pairs of subunits - P2X2 and P2X4, and P2X4 and P2X7. EXPERIMENTAL APPROACH We used several experimental approaches, including in situ proximity ligation, co-immunoprecipitation, co-isolation on affinity beads, chemical cross-linking and atomic force microscopy (AFM) imaging. KEY RESULTS Both pairs of subunits co-localize upon co-transfection, interact intimately within cells, and can be co-immunoprecipitated and co-isolated from cell extracts. Despite this, chemical cross-linking failed to show evidence for heteromer formation. AFM imaging of isolated receptors showed that all three subunits had the propensity to form receptor dimers. This self-association is likely to account for the observed close interaction between the subunit pairs...

Alteration of purinergic P2X(4) and P2X(7) receptor expression in rats with temporal-lobe epilepsy induced by pilocarpine

DONA, Flavia; ULRICH, Henning; PERSIKE, Daniele Suzete; CONCEICAO, Isaltino Marcelo; BLINI, Joao Paulo; CAVALHEIRO, Esper Abrao; FERNANDES, Maria Jose Silva
Fonte: ELSEVIER SCIENCE BV Publicador: ELSEVIER SCIENCE BV
Tipo: Artigo de Revista Científica
Português
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Although ATP and P2X receptor activity have been lately associated with epilepsy, little is known regarding their exact roles in epileptogenesis. Temporal-lobe epilepsy (TLE) in rat was induced by pilocarpine in order to study changes of hippocampal P2X(2), P2X(4) and P2X(7) receptor expression during acute, latent or chronic phases of epilepsy. During acute and chronic phases increased P2X(7) receptor expression was principally observed in glial cells and glutamatergic nerve terminals, suggesting participation of this receptor in the activation of inflammatory and excitotoxic processes during epileptogenesis. No significant alterations of hippocampal P2X(2) and P2X(4) receptor expression was noted during the acute or latent phase when compared to the control group, indicating that these receptors are not directly involved with the initiation of epilepsy. However, the reduction of hippocampal P2X(4) receptor immunostaining in the chronic phase could reflect neuronal toss or decreased GABAergic signaling. (C) 2008 Elsevier B.V. All rights reserved.; FAPESP (Fundacao de Amparo a Pesquisa do Estado de Sao Paulo); Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP); CNPq (Conselho Nacional de Desenvolvimento Cientifico e Tecnologico); Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq); Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES); CAPES (Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior)...

Regulation of neurogenesis and gliogenesis of retinoic acid-induced P19 embryonal carcinoma cells by P2X2 and P2X7 receptors studied by RNA interference

Yuahasi, Katia Kioko; Demasi, Marcos Angelo Almeida; Tamajusuku, Alessandra S. K.; Lenz, Guido; Sogayar, Mari Cleide; Fornazari, Maynara; Lameu, Claudiana; Nascimento, Isis C.; Glaser, Talita; Schwindt , Telma Tiemi; Negraes, Priscilla D; Ulrich, Henning
Fonte: PERGAMON-ELSEVIER SCIENCE LTD; OXFORD Publicador: PERGAMON-ELSEVIER SCIENCE LTD; OXFORD
Tipo: Artigo de Revista Científica
Português
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Embryonic carcinoma cells are widely used models for studying the mechanisms of proliferation and differentiation occurring during early embryogenesis. We have now investigated how down-regulation of P2X2 and P2X7 receptor expression by RNA interference (RNAi) affects neural differentiation and phenotype specification of P19 embryonal carcinoma cells. Wild-type P19 embryonal carcinoma cells or cells stably expressing shRNAs targeting P2X2 or P2X7 receptor expression were induced to differentiate into neurons and glial cells in the presence of retinoic acid. Silencing of P2X2 receptor expression along differentiation promoted cell proliferation and an increase in the percentage of cells expressing glial-specific GFAP, while the presence of beta-3 tubulin-positive cells diminished at the same time. Proliferation induction in the presence of stable anti-P2X2 receptor RNAi points at a mechanism where glial proliferation is favored over growth arrest of progenitor cells which would allow neuronal maturation. Differently from the P2X2 receptor, inhibition of P2X7 receptor expression during neural differentiation of P19 cells resulted in a decrease in cell proliferation and GFAP expression, suggesting the need of functional P2X7 receptors for the progress of gliogenesis. The results obtained in this study indicate the importance of purinergic signaling for cell fate determination during neural differentiation...

Modulation of intercellular communication in macrophages: possible interactions between GAP junctions and P2 receptors

Fortes, F. S. A.; Pecora, Iracy Lea; Persechini, P. M.; Hurtado, S.; Costa, V; Coutinho-Silva, R.; Braga, MBM; Silva-Filho, F. C.; Bisaggio, R. C.; Farias, F. P. de; Scemes, E.; Carvalho, A. C. C. de; Goldenberg, R. C. S.
Fonte: Company of Biologists Ltd Publicador: Company of Biologists Ltd
Tipo: Artigo de Revista Científica Formato: 4717-4726
Português
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66.21%
Gap junctions are connexin-formed channels that play an important role in intercellular communication in most cell types. In the immune system, specifically in macrophages, the expression of connexins and the establishment of functional gap junctions are still controversial issues. Macrophages express P2X(7) receptors that, once activated by the binding of extracellular ATP, lead to the opening of transmembrane pores permeable to molecules of up to 900 Da. There is evidence suggesting an interplay between gap junctions and P2 receptors in different cell systems. Thus, we used ATP-sensitive and -insensitive J774.G8 macrophage cell lines to investigate this interplay. To study junctional communication in J774-macrophage-like cells, we assessed cell-to-cell communication by microinjecting Lucifer Yellow. Confluent cultures of ATP-sensitive J774 cells (ATP-s cells) are coupled, whereas ATP-insensitive J774 cells (ATP-i cells), derived by overexposing J774 cells to extracellular ATP until they do not display the phenomenon of ATP-induced permeabilization, are essentially uncoupled. Western-blot and reverse-transcription polymerase chain reaction assays revealed that ATP-s and ATP-i cells express connexin43 (Cx43), whereas only ATP-s cells express the P2X(7) receptor. Accordingly...

Immunohistochemical identification of cells expressing ATP-gated cation channels (P2X receptors) in the adult rat thyroid

GLASS, RAINER; BURNSTOCK, GEOFFREY
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /05/2001 Português
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We carried out immunohistochemistry and western blotting of fresh frozen sections and crude extracts from adult rat thyroids. The histochemical and immunoblotting studies were performed with P2X receptor antibodies from 2 different sources. P2X-immunopositive cells were identified by fluorescence double labelling and confocal microscopy. Results of the western blotting experiments showed double bands of approximately 70 kDa and 140 kDa for all 7 P2X receptor subtypes with both sets of antibodies. Histochemical stains with antibodies from both sources also gave essentially identical results. P2X1, P2X2 and P2X6 receptors were detected exclusively in vascular smooth muscle; P2X5 and P2X7 receptors were also present on vascular smooth muscle. Endothelial cells stained for P2X3, P2X4 and P2X7 receptors. Thyroid follicular cells displayed immunoreactivity for P2X3, P2X4 and P2X5 receptors. No immunostaining for P2X receptors was observed on C-cells. Possible roles for the broad expression of P2X receptor subtypes in the rat thyroid are discussed.

P2X receptor expression in mouse urinary bladder and the requirement of P2X1 receptors for functional P2X receptor responses in the mouse urinary bladder smooth muscle

Vial, C; Evans, R J
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /12/2000 Português
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We have used subtype selective P2X receptor antibodies to determine the expression of P2X1–7 receptor subunits in the mouse urinary bladder. In addition we have compared P2X receptor mediated responses in normal and P2X1 receptor deficient mice to determine the contribution of the P2X1 receptor to the mouse bladder smooth muscle P2X receptor phenotype.P2X1 receptor immunoreactivity was restricted to smooth muscle of the bladder and arteries and was predominantly associated with the extracellular membrane. Diffuse P2X2 and P2X4 receptor immunoreactivity not associated with the extracellular membrane was detected in the smooth muscle and epithelial layers. Immunoreactivity for the P2X7 receptor was associated with the innermost epithelial layers and some diffuse staining was seen in the smooth muscle layer. P2X3, P2X5 and P2X6 receptor immunoreactivity was not detected.P2X receptor mediated inward currents and contractions were abolished in bladder smooth muscle from P2X1 receptor deficient mice. In normal bladder nerve stimulation evoked contractions with P2X and muscarinic acetylcholine (mACh) receptor mediated components. In bladder from the P2X1 receptor deficient mouse the contraction was mediated solely by mACh receptors. Contractions to carbachol were unaffected in P2X1 receptor deficient mice demonstrating that there had been no compensatory effect on mACh receptors.These results indicate that homomeric P2X1 receptors underlie the bladder smooth muscle P2X receptor phenotype and suggest that mouse bladder from P2X1 receptor deficient and normal animals may be models of human bladder function in normal and diseased states.

Local regulation of [3H]-noradrenaline release from the isolated guinea-pig right atrium by P2X-receptors located on axon terminals

Sperlágh, Beáta; Erdélyi, Ferenc; Szabó, Gábor; Vizi, E Sylvester
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /12/2000 Português
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In this study the regulation of cardiac sympathetic outflow by presynaptic P2X receptor-gated ion channels was examined.ATP (30 μM–1 mM) and other P2-receptor agonists elicited [3H]-noradrenaline ([3H]-NA) outflow from the isolated guinea-pig right atrium with the potency order of ATP>2-methyl-thioATP>α,β-methylene-ATP=ADP, whereas β,γ-methylene-L-ATP was inactive.Ca2+-free conditions abolished both electrical field stimulation (EFS)- and ATP-evoked release of tritium. Unlike from EFS-induced outflow, ATP-induced [3H]-NA outflow was not reduced by ω-Conotoxin-GVIA (100 nM), Cd2+ (100 μM) and tetrodotoxin (1 μM).The rapid extracellular decomposition of ATP was revealed by HPLC analysis. However, the effect of ATP to promote [3H]-NA release was not prevented by 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 250 nM), 3,7-dimethyl-1-propargylxanthine (DMPX, 250 nM), or by reactive blue 2 (RB2, 10 μM), antagonists of A1-, A2- and inhibitory P2 receptors.Zn2+ (50 μM), the P2X-receptor modulator potentiated, and P2X receptor antagonists, i.e. suramin (300 μM), pyridoxal-phosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS, 30 μM) and 2′-o-(trinitrophenyl)-adenosine 5′-triphosphate (TNP-ATP, 30 μM) antagonized the ATP (1 mM)-evoked response.RT–PCR study revealed the expression of P2X2 and P2X3 receptor mRNAs in guinea-pig superior cervical ganglion.PPADS (30 μM) significantly reduced the EFS-induced [3H]-NA outflow in the presence DPCPX (250 nM) and RB2 (10 μM).In summary a P2X-type purinoceptor regulates noradrenaline release from the isolated right atrium of the guinea-pig. The pharmacological profile of the receptor resemble to homo-oligomeric P2X3 or hetero-oligomeric P2X2/P2X3 complexes...

Nerve evoked P2X receptor contractions of rat mesenteric arteries; dependence on vessel size and lack of role of L-type calcium channels and calcium induced calcium release

Gitterman, D P; Evans, R J
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /03/2001 Português
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26.33%
Contractile responses to short trains of nerve stimulation have been characterized in small, medium and large arteries from the rat mesenteric circulation (5th – 6th, 2nd – 3rd and 1st order, respectively). In addition, sources of calcium for smooth muscle contraction have been investigated.Nerve stimulation (10 pulses at 10 Hz) evoked reproducible contractions. The P2 receptor antagonist suramin (100 μM) reduced constrictions by 65.3±7.4, 82.7±3.3 and 3.1±6.1% in small, medium and large arteries respectively. The α-adrenoceptor antagonist prazosin (0.1 μM) reduced responses by 32.6±2.6, 27.0±1.5 and 97.0±1.9% respectively.The L-type calcium channel antagonist nifedipine (1 μM) reduced nerve-evoked contractions by 2.8±3.3, 10.0±3.7 and 13.5±2.7% in small, medium and large arteries respectively. When the adrenergic component of contraction was blocked by prazosin (0.1 μM) nifedipine reduced responses by 4.6±7.9, 14.3±2.0 and 3.0±1.9% respectively.Contractile responses to exogenous α,β-meATP were unaffected by the depletion of calcium stores with cyclopiazonic acid (30 μM). This indicates that mobilization of calcium from internal stores is not required for P2X receptor mediated smooth muscle contraction.We conclude that for neurogenic responses...

Functional evidence that ATP or a related purine is an inhibitory NANC neurotransmitter in the mouse jejunum: study on the identity of P2X and P2Y purinoceptors involved

De Man, Joris G; De Winter, Benedicte Y; Seerden, Tom C; De Schepper, Heiko U; Herman, Arnold G; Pelckmans, Paul A
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Português
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36.24%
Conflicting views exist on whether ATP is a neurotransmitter in the enteric nervous system. We investigated the role of ATP in enteric transmission in circular muscle strips of the mouse jejunum.On PGF2α-precontracted muscle strips and in the presence of atropine and guanethidine, electrical field stimulation (EFS, 1–8 Hz) of nonadrenergic noncholinergic (NANC) nerves induced transient relaxations that were abolished by the nerve-conductance blocker tetrodotoxin. The NO synthase blocker L-nitroarginine (L-NOARG) partially inhibited the NANC relaxations to EFS, but fast-twitch relaxations to EFS were still observed in the presence of L-NOARG.In the presence of L-NOARG, ATP, the P2X receptor agonist αβMeATP and the P2Y receptor agonist ADPβS relaxed jejunal muscle strips. Tetrodotoxin did not affect the relaxation to ATP and ADPβS, but inhibited that to αβMeATP.The L-NOARG-resistant NANC relaxations to EFS were almost abolished by apamin, a blocker of small-conductance Ca2+ activated K+ channels, and by suramin and PPADS, blockers of P2 purinoceptors. Relaxations to ATP were almost abolished by apamin and suramin but not affected by PPADS.Desensitisation of αβMeATP-sensitive P2X receptors, the P2X receptor blocker Evans blue and the P2X1...

Effects of diadenosine polyphosphates (ApnAs) and adenosine polyphospho guanosines (ApnGs) on rat mesenteric artery P2X receptor ion channels

Lewis, C J; Gitterman, D P; Schlüter, H; Evans, R J
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /01/2000 Português
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26.32%
Diadenosine polyphosphates (ApnAs, n=3–7) and adenosine polyphospho guanosines (ApnGs, n=3–6) are naturally occurring vasoconstrictor substances found in platelets. These vasoconstrictor actions are thought to be mediated through the activation of P2X receptors for ATP. The effects of ApnAs and ApnGs at P2X receptors on rat mesenteric arteries were determined in contraction studies and using the patch clamp technique on acutely dissociated artery smooth muscle cells.P2X1 receptor immunoreactivity was detected in the smooth muscle layer of artery rings. The sensitivity to α,β-methylene ATP and desensitizing nature of rat mesenteric artery P2X receptors correspond closely to those of recombinant P2X1 receptors.Ap4A, Ap5A and Ap6A evoked concentration dependent P2X receptor inward currents which desensitized during the application of higher concentrations of agonist. The agonist order of potency was Ap5A⩾Ap6A⩾Ap4A>>Ap3A. Ap2A and Ap7A were ineffective. Similar results were obtained in contraction studies except for Ap7A which evoked a substantial contraction.ApnGs (n=2–6)(30 μM) evoked P2X receptor inward currents in mesenteric artery smooth muscle cells. ApnGs (n=4–6) were less effective than the corresponding ApnA.This study shows that at physiologically relevant concentrations ApnAs and ApnGs can mediate contraction of rat mesenteric arteries through the activation of P2X1-like receptors. However the activity of the longer chain polyphosphates (n=6–7) may be overestimated in whole tissue studies due to metabolic breakdown to yield the P2X receptor agonists ATP and adenosine tetraphosphate.

Mutual occlusion of P2X ATP receptors and nicotinic receptors on sympathetic neurons of the guinea-pig

Searl, T J; Redman, R S; Silinsky, E M
Fonte: Blackwell Science Inc Publicador: Blackwell Science Inc
Tipo: Artigo de Revista Científica
Publicado em 01/08/1998 Português
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The interaction of ion channels activated by nicotinic receptor agonists with ion channels gated by extracellular ATP (i.e. P2X receptors) was studied on sympathetic neurons acutely dissociated from coeliac ganglia of the guinea-pig. Patch clamp methods were used to measure the inward current generated through these non-selective cationic channels under voltage clamp.At the whole cell level, the specific nicotinic receptor agonists nicotine (5-100 μm) or cytisine (50-75 μm) and the P2X receptor agonists ATP (0.1-7 μm) or α,β-methylene ATP (6 μm) were examined separately and in the presence of the other receptor activator. When a nicotinic and P2X receptor agonist were applied together, mutually occlusive effects were generally observed. This occurred even with concentrations of agonists that in themselves generated little to no inward current.The occlusive effects of nicotinic agonists on ATP-gated currents were blocked by the nicotinic receptor/ion channel blocker hexamethonium (150 μm). The occlusive effects of ATP analogues on inward currents generated by nicotinic agonists were blocked by the P2X receptor antagonist suramin (100 μm).Mutual occlusion of the effects of nicotinic agonists and ATP analogues were also observed when currents through single channels were studied in excised (outside-out) patches.The results suggest that nicotinic receptors and P2X ATP receptors do not act independently in these sympathetic neurons.

Cloning and Characterization of a P2X Receptor Expressed in the Central Nervous System of Lymnaea stagnalis

Bavan, Selvan; Straub, Volko A.; Webb, Tania E.; Ennion, Steven J.
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 29/11/2012 Português
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26.25%
P2X receptors are membrane ion channels gated by extracellular ATP. Mammals possess seven distinct P2X subtypes (P2X1-7) that have important functions in a wide array of physiological processes including roles in the central nervous system (CNS) where they have been linked to modulation of neurotransmitter release. We report here the cloning and functional characterization of a P2X receptor from the mollusc Lymnaea stagnalis. This model organism has a relatively simple CNS consisting of large readily identifiable neurones, a feature which together with a well characterized neuronal circuitry for important physiological processes such as feeding and respiration makes it an attractive potential model to examine P2X function. Using CODEHOP PCR we identified a single P2X receptor (LymP2X) in Lymnaea CNS which was subsequently cloned by RT-PCR. When heterologously expressed in Xenopus oocytes, LymP2X exhibited ATP evoked inward currents (EC50 6.2 µM) which decayed during the continued presence of agonist. UTP and ADP did not activate the receptor whereas αβmeATP was a weak agonist. BzATP was a partial agonist with an EC50 of 2.4 µM and a maximal response 33% smaller than that of ATP. The general P2 receptor antagonists PPADS and suramin both inhibited LymP2X currents with IC50 values of 8.1 and 27.4 µM respectively. LymP2X is inhibited by acidic pH whereas Zn2+ and Cu2+ ions exhibited a biphasic effect...

Distribution of purinergic P2X receptors in the equine digit, cervical spinal cord and dorsal root ganglia

Zamboulis, D. E.; Senior, J. M.; Clegg, P. D.; Gallagher, J. A.; Carter, S. D.; Milner, P. I.
Fonte: Springer Netherlands Publicador: Springer Netherlands
Tipo: Artigo de Revista Científica
Português
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36.32%
Purinergic pathways are considered important in pain transmission, and P2X receptors are a key part of this system which has received little attention in the horse. The aim of this study was to identify and characterise the distribution of P2X receptor subtypes in the equine digit and associated vasculature and nervous tissue, including peripheral nerves, dorsal root ganglia and cervical spinal cord, using PCR, Western blot analysis and immunohistochemistry. mRNA signal for most of the tested P2X receptor subunits (P2X1–5, 7) was detected in all sampled equine tissues, whereas P2X6 receptor subunit was predominantly expressed in the dorsal root ganglia and spinal cord. Western blot analysis validated the specificity of P2X1–3, 7 antibodies, and these were used in immunohistochemistry studies. P2X1–3, 7 receptor subunits were found in smooth muscle cells in the palmar digital artery and vein with the exception of the P2X3 subunit that was present only in the vein. However, endothelial cells in the palmar digital artery and vein were positive only for P2X2 and P2X3 receptor subunits. Neurons and nerve fibres in the peripheral and central nervous system were positive for P2X1–3 receptor subunits, whereas glial cells were positive for P2X7 and P2X1 and 2 receptor subunits. This previously unreported distribution of P2X subtypes may suggest important tissue specific roles in physiological and pathological processes.

Gintonin, a Ginseng-Derived Lysophosphatidic Acid Receptor Ligand, Potentiates ATP-Gated P2X1 Receptor Channel Currents

Choi, Sun-Hye; Kim, Hyeon-Joong; Kim, Bo-Ra; Shin, Tae-Joon; Hwang, Sung-Hee; Lee, Byung-Hwan; Lee, Sang-Mok; Rhim, Hyewhon; Nah, Seung-Yeol
Fonte: Korea Society for Molecular and Cellular Biology Publicador: Korea Society for Molecular and Cellular Biology
Tipo: Artigo de Revista Científica
Português
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35.93%
Ginseng, the root of Panax ginseng C.A. Meyer, is used as a general tonic. Recently, we isolated a novel ginseng-derived lysophosphatidic acid (LPA) receptor ligand, gintonin. Gintonin activates G protein-coupled LPA receptors with high affinity in cells endogenously expressing LPA receptors, e.g., Xenopus oocytes. P2X receptors are ligand-gated ion channels activated by extracellular ATP, and 7 receptor subtypes (P2X1–P2X7) have been identified. Most of the P2X1 receptors are expressed in the smooth muscles of genitourinary organs involved in reproduction. A main characteristic of the P2X1 receptor is rapid desensitization after repeated ATP treatment of cells or tissues expressing P2X1 receptors. In the present study, we examined the effect of gintonin on P2X1 receptor channel activity. P2X1 receptors were heterologously expressed in Xenopus oocytes. ATP treatment of oocytes expressing P2X1 receptors induced large inward currents (IATP), but repetitive ATP treatments induced a rapid desensitization of IATP. Gintonin treatment after P2X1 receptor desensitization potentiated IATP in a concentration-dependent manner. We further examined the signaling transduction pathways involved in gintonin-mediated potentiation of IATP. Gintonin-mediated IATP potentiation was blocked by Ki16425...

P2X purinoceptor-induced sensitization of ferret vagal mechanoreceptors in oesophageal inflammation

Page, A.; O'Donnell, T.; Blackshaw, L.
Fonte: Blackwell Publishing Ltd Publicador: Blackwell Publishing Ltd
Tipo: Artigo de Revista Científica
Publicado em //2000 Português
Relevância na Pesquisa
36%
1. Using an in vitro single unit recording technique we studied the changes in mechanical and chemical sensitivity of vagal afferent fibres in acute oesophagitis, with particular attention to inflammatory products such as purines. 2. Histologically verified oesophagitis was induced by oesophageal perfusion of 1 mg ml-1 pepsin in 150 mM HCl in anaesthetized ferrets for 30 min on two consecutive days. Controls were infused with 154 mM NaCl. 3. The number of action potentials evoked in oesophageal mucosal afferents by mucosal stroking with calibrated von Frey hairs (10-1000 mg) was stimulus dependent. In oesophagitis responsiveness was reduced across the range of stimuli compared with controls. 4. Topical application of the P2X purinoceptor agonist alphabeta-methylene ATP had no direct excitatory effect on afferents. In oesophagitis, but not in controls, there was a significant increase in responses to stroking with von Frey hairs during superfusion with alphabeta-methylene ATP (1 microM). 5. Mucosal afferents responded directly to one or more chemical stimuli: 26 % (5/19 afferents) responded in controls, and 47 % (7/15 afferents) in oesophagitis. There were no differences in responsiveness to bradykinin (1 microM), prostaglandin E2 (100 microM)...

P2X7 receptors activate protein kinase D and p42/p44 mitogen-activated protein kinase (MAPK) downstream of protein kinase C.

Bradford, Michelle D; Soltoff, Stephen P
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 15/09/2002 Português
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Protein kinase D (PKD), also called protein kinase Cmu (PKCmu), is a serine/threonine kinase that has unique enzymic and structural properties distinct from members of the PKC family of proteins. In freshly isolated rat parotid acinar salivary cells, extracellular ATP rapidly increased the activity and phosphorylation of PKD. The stimulation by ATP required high concentrations, was mimicked by the P2X(7) receptor ligand BzATP [2'- and 3'-O-(4-benzoylbenzoyl)ATP], and was blocked by Mg(2+) and 4,4'-di-isothiocyano-2,2'-stilbene disulphonate (DIDS), suggesting that activation of PKD was mediated by P2X(7) receptors, which are ligand-gated non-selective cation channels. Phorbol ester (PMA) and the activation of muscarinic and substance P receptors also increased PKD activity. PKC inhibitors blocked ligand-dependent PKD activation and phosphorylation, determined by in vitro phosphorylation studies and by phospho-specific antibodies to two activation loop sites (Ser(744) and Ser(748)) and an autophosphorylation site (Ser(916)). ATP and BzATP also increased the tyrosine phosphorylation and activity of PKCdelta, and these stimuli also increased extracellular signal-regulated protein kinase (ERK) 1/2 activity in a PKC-dependent manner. PKD activation was not promoted by pervanadate (an inhibitor of tyrosine phosphatases) and was not blocked by PP1 (an inhibitor of Src family kinases) or genistein (a tyrosine kinase inhibitor)...

P2X purinoceptors in cultured myenteric neurons of guinea-pig small intestine.

Zhou, X; Galligan, J J
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 01/11/1996 Português
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26.26%
1. Fast excitatory postsynaptic currents (fEPSCs) and responses to exogenously applied purinoceptor agonists were studied in primary cultures of myenteric neurons from guinea-pig small intestine. Whole-cell and outside-out configurations of the patch clamp technique were used. Hexamethonium (100 microM) partly inhibited fEPSCs in 28% of neurons. Hexamethonium-resistant fEPSCs were inhibited by 97 +/- 2% by the P2X receptor antagonist, pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS, 10 microM). 2. ATP caused two types of inward currents. In 92% of neurons (n = 123), ATP caused a slowly desensitizing current that declined with a double exponential time course (tau 1 = 7.1 +/- 2.0 s; tau 2 = 57 +/- 7.4 s, n = 4). The rank order potency for purinoceptor agonists in these neurons was ATP > 2-methylthio-ATP (2-MeSATP) > > alpha, beta-methylene ATP (alpha, beta-me ATP) > beta, gamma-meATP > ADP. The EC50 values for ATP and 2-MeSATP were 40 and 65 microM, respectively. alpha, beta-MeATP acted as a partial agonist at these receptors. In 8% of neurons (n = 11), ATP-induced currents desensitized rapidly with a double exponential time course (tau 1 = 0.13 +/- 0.015 s; tau 2 = 2.2 +/- 1.3 s, n = 4); alpha, beta-meATP caused similar responses in these cells. Both types of ATP-induced current were associated with an increased conductance and an inwardly rectifying I-V relationship (Erev = 10 mV). Halving [Na+]o shifted the reversal potential of ATP currents by -22 +/- 6 mV. 3. ATP activated single channel currents in outside-out patches. The single channel I-V relationship was linear between -120 and 60 mV (Erev approximately 0 mV). Single channel conductance between -100 and -60 mV was 25 +/- 2 pS. Single channel open probability was voltage dependent and decreased from 0.05 +/- 0.01 at -100 mV to 0.007 +/- 0.002 at +40 mV. 4. These data show that P2X purinoceptors mediate some fEPSCs in cultured myenteric neurons. Myenteric neurons express the fast-desensitizing alpha...