共查询到20条相似文献,搜索用时 0 毫秒
1.
Kenji F Shoji Pablo J Sáez Paloma A Harcha Hector L Aguila Juan C Sáez 《Channels (Austin, Tex.)》2014,8(2):142-156
Death of murine T cells induced by extracellular ATP is mainly triggered by activation of purinergic P2X7 receptors (P2X7Rs). However, a link between P2X7Rs and pannexin1 (Panx1) channels, which are non-selective, has been recently demonstrated in other cell types. In this work, we characterized the expression and cellular distribution of pannexin family members (Panxs 1, 2 and 3) in isolated T cells. Panx1 was the main pannexin family member clearly detected in both helper (CD4+) and cytotoxic (CD8+) T cells, whereas low levels of Panx2 were found in both T-cell subsets. Using pharmacological and genetic approaches, Panx1 channels were found to mediate most ATP-induced ethidium uptake since this was drastically reduced by Panx1 channel blockers (10Panx1, Probenecid and low carbenoxolone concentration) and absent in T cells derived from Panx1−/− mice. Moreover, electrophysiological measurements in wild-type CD4+ cells treated with ATP unitary current events and pharmacological sensitivity compatible with Panx1 channels were found. In addition, ATP release from T cells treated with 4Br-, a calcium ionophore, was completely blocked with inhibitors of both connexin hemichannels and Panx1 channels. Panx1 channel blockers drastically reduced the ATP-induced T-cell mortality, indicating that Panx1 channels mediate the ATP-induced T-cell death. However, mortality was not reduced in T cells of Panx1−/− mice, in which levels of P2X7Rs and ATP-induced intracellular free Ca2+ responses were enhanced suggesting that P2X7Rs take over Panx1 channels lose-function in mediating the onset of cell death induced by extracellular ATP. A23187相似文献
2.
The purinergic receptor P2X(7) is part of a complex signaling mechanism participating in a variety of physiological and pathological processes. Depending on the activation scheme, P2X(7) receptors in vivo are non-selective cation channels or form large pores that can mediate apoptotic cell death. Expression of P2X(7)R in Xenopus oocytes results exclusively in formation of a non-selective cation channel. However, here we show that co-expression of P2X(7)R with pannexin1 in oocytes leads to the complex response seen in many mammalian cells, including cell death with prolonged ATP application. While the cation channel activity is resistant to carbenoxolone treatment, this gap junction and hemichannel blocking drug suppressed the currents induced by ATP in pannexin1/P2X(7)R co-expressing cells. Thus, pannexin1 appears to be the molecular substrate for the permeabilization pore (or death receptor channel) recruited into the P2X(7)R signaling complex. 相似文献
3.
Daniela Gentile Mariarita Natale Pietro Enea Lazzerini Pier Leopoldo Capecchi Franco Laghi-Pasini 《Purinergic signalling》2015,11(4):435-440
Many previous studies have demonstrated that P2X7 receptors (P2X7Rs) have a pleiotropic function in different pathological conditions and could represent a novel target for the treatment of a range of diseases. In particular, recent studies have explored the role of P2X7R in fibrosis, the pathological outcome of most chronic inflammatory diseases. The aim of this review is to discuss the biological features of P2X7R and summarize the current knowledge about the putative role of the P2X7R in triggering fibrosis in a wide spectrum of organs such as the lung, kidney, liver, pancreas, and heart. 相似文献
4.
The tetra-anionic form of ATP (ATP4-) is known to induce monovalent and divalent ion fluxes in cells that express purinergic P2X7 receptors and with sustained application of ATP it has been shown that dyes as large as 831 Da can permeate the cell membrane. The current study explores the kinetics of loading alpha,alpha-trehalose (342 Da) into ATP stimulated J774.A1 cells, which are known to express the purinergic P2X7 receptor. Cells that were incubated at 37 degrees C in a 50 mM phosphate buffer (pH 7.0) containing 225 mM trehalose and 5 mM ATP, were shown to load trehalose linearly over time. Concentrations of approximately 50 mM were reached within 90 min of incubation. Cells incubated in the same solution at 4 degrees C loaded minimally, consistent with the inactivity of the receptor at low temperatures. However, extended incubation at 37 degrees C (>60 min) resulted in zero next-day survival, with adverse effects appearing even with incubation periods as short as 30 min. By using a two-step protocol with a short time period at 37 degrees C to allow pore formation, followed by an extended loading period on ice, cells could be loaded with up to 50 mM trehalose while maintaining good next day recovery (49 +/- 12% by Trypan blue exclusion, 56 +/- 20% by alamarBlue assay). Cells porated by this method and allowed an overnight recovery period exhibited improved dehydration tolerance suggesting a role for ATP poration in the anhydrous preservation of cells. 相似文献
5.
P2X3 and P2X2+3 receptors are present on sensory neurons, where they contribute not only to transient nociceptive responses, but also to
hypersensitivity underlying pathological pain states elicited by nerve injuries. Increased signalling through P2X3 and P2X2+3 receptors may arise from an increased routing to the plasma membrane and/or gain of function of pre-existing receptors. An
obvious effector mechanism for functional modulation is protein kinase C (PKC)-mediated phosphorylation, since all P2X family
members share a conserved consensus sequence for PKC, TXR/K, within the intracellularly located N-terminal domain. Contradictory
reports have been published regarding the exact role of this motif. In the present study, we confirm that site-directed elimination
of the potential phosphor-acceptor threonine or the basic residue in the P+2 position of the TXR/K sequence accelerates desensitization
of P2X2 receptors and abolishes P2X3 receptor function. Moreover, the PKC activator phorbol 12-myristate 13-acetate increased P2X3 (but not P2X2) receptor-mediated currents. Biochemically, however, we were unable to demonstrate by various experimental approaches a direct
phosphorylation of wild-type P2X2 and P2X3 receptors expressed in both Xenopus laevis oocytes and HEK293 cells. In conclusion, our data support the view that the TXR/K motif plays an important role in P2X function
and that phorbol 12-myristate 13-acetate is capable of modulating some P2X receptor subtypes. The underlying mechanism, however,
is unlikely to involve direct PKC-mediated P2X receptor phosphorylation. 相似文献
6.
Lu H Burns D Garnier P Wei G Zhu K Ying W 《Biochemical and biophysical research communications》2007,362(4):946-950
NADH plays critical roles in mitochondrial functions and energy metabolism. There has been no study demonstrating that NADH can be transported across the plasma membranes of cells. In this study we tested our hypothesis that NADH can be transported across the plasma membranes of astrocytes by a P2X7 receptor (P2X7R)-mediated mechanism. We found that treatment of astrocytes with NADH led to increases in both intracellular NADH and NAD+. Three lines of studies suggest that P2X7R mediates the NADH transport into astrocytes: the P2X receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) blocked the NADH transport; RNAi knockdown of P2X7R led to decreased NADH transport; and transfection of HEK293 cells with mouse P2X7R cDNA led to increased NADH transport. Collectively, our study provides the first direct evidence demonstrating that NADH can be transported across the plasma membranes of astrocytes by a P2X7R-mediated mechanism. Our study also suggests a novel approach for manipulating intracellular NADH and NAD+ levels. 相似文献
7.
Identification and characterization of splice variants of the human P2X7 ATP channel 总被引:9,自引:0,他引:9
Cheewatrakoolpong B Gilchrest H Anthes JC Greenfeder S 《Biochemical and biophysical research communications》2005,332(1):17-27
The P2X7 channel is a member of the P2X family of ligand-gated ion channels which respond to ATP as the endogenous agonist. Studies suggest that P2X7 has a potentially pivotal role in inflammatory responses largely stemming from its role in mediating the release of IL-1beta in response to ATP. We report the identification of seven variants of human P2X7 which result from alternative splicing. Two of these variants (one lacking the first transmembrane domain, the second lacking the entire cytoplasmic tail) were compared to the full-length channel. Real-time PCR analysis demonstrated that both variants were expressed in various tissues and that the cytoplasmic tail deleted variant is highly expressed. Deletion of the first transmembrane domain resulted in a non-functional channel. Deletion of the cytoplasmic tail did not affect ion movement but severely affected the ability to form a large pore and to induce activation of caspases. 相似文献
8.
Perez-Medrano A Donnelly-Roberts DL Florjancic AS Nelson DW Li T Namovic MT Peddi S Faltynek CR Jarvis MF Carroll WA 《Bioorganic & medicinal chemistry letters》2011,21(11):3297-3300
Synthesis and biological evaluation of a novel class of substituted N-benzyl-1-(2,3-dichlorophenyl)-1H-tetrazol-5-amine derivatives resulted in the identification of potent P2X7 antagonists. These compounds were assayed for activity at both the human and rat P2X7 receptors. On the benzyl moiety, a variety of functional groups were tolerated, including both electron-withdrawing and electron-donating substituents. Ortho-substitution on the benzyl group provided the greatest potency. The ortho-substituted analogs showed approximately 2.5-fold greater potency at human compared to rat P2X7 receptors. Compounds 12 and 38 displayed hP2X7pIC50s >7.8 with less than 2-fold difference in potency at the rP2X7. 相似文献
9.
Kong Q Wang M Liao Z Camden JM Yu S Simonyi A Sun GY Gonzalez FA Erb L Seye CI Weisman GA 《Purinergic signalling》2005,1(4):337-347
Apoptosis is a major cause of cell death in the nervous system. It plays a role in embryonic and early postnatal brain development
and contributes to the pathology of neurodegenerative diseases. Here, we report that activation of the P2X7 nucleotide receptor (P2X7R) in rat primary cortical neurons (rPCNs) causes biochemical (i.e., caspase activation) and morphological (i.e., nuclear
condensation and DNA fragmentation) changes characteristic of apoptotic cell death. Caspase-3 activation and DNA fragmentation
in rPCNs induced by the P2X7R agonist BzATP were inhibited by the P2X7R antagonist oxidized ATP (oATP) or by pre-treatment of cells with P2X7R antisense oligonucleotide indicating a direct involvement of the P2X7R in nucleotide-induced neuronal cell death. Moreover, Z-DEVD-FMK, a specific and irreversible cell permeable inhibitor of
caspase-3, prevented BzATP-induced apoptosis in rPCNs. In addition, a specific caspase-8 inhibitor, Ac-IETD-CHO, significantly
attenuated BzATP-induced caspase-9 and caspase-3 activation, suggesting that P2X7R-mediated apoptosis in rPCNs occurs primarily through an intrinsic caspase-8/9/3 activation pathway. BzATP also induced the
activation of C-jun N-terminal kinase 1 (JNK1) and extracellular signal-regulated kinases (ERK1/2) in rPCNs, and pharmacological
inhibition of either JNK1 or ERK1/2 significantly reduced caspase activation by BzATP. Taken together, these data indicate
that extracellular nucleotides mediate neuronal apoptosis through activation of P2X7Rs and their downstream signaling pathways involving JNK1, ERK and caspases 8/9/3. 相似文献
10.
Sluyter R Shemon AN Wiley JS 《Biochemical and biophysical research communications》2007,355(1):169-173
Activation of cation channels causes erythrocyte phosphatidylserine (PS) exposure and cell shrinkage. Human erythrocytes express the P2X7 receptor, an ATP-gated cation channel. The two most potent P2X7 agonists, BzATP and ATP, stimulated PS exposure in human erythrocytes. Other nucleotides also induced erythrocyte PS exposure with an order of agonist potency of BzATP > ATP > 2MeSATP > ATPγS; however neither ADP nor UTP had an effect. ATP induced PS exposure in erythrocytes in a dose-dependent fashion with an EC50 of ∼75 μM. BzATP- and ATP-induced erythrocyte PS exposure was impaired by oxidised ATP, as well as in erythrocytes from subjects who had inherited loss-of-function polymorphisms in the P2X7 receptor. ATP-induced PS exposure in erythrocytes was not significantly altered in the presence of EGTA excluding a role for extracellular Ca2+. These results show that P2X7 activation by extracellular ATP can induce PS exposure in erythrocytes. 相似文献
11.
Matasi JJ Brumfield S Tulshian D Czarnecki M Greenlee W Garlisi CG Qiu H Devito K Chen SC Sun Y Bertorelli R Geiss W Le VD Martin GS Vellekoop SA Haber J Allard ML 《Bioorganic & medicinal chemistry letters》2011,21(12):3805-3808
Structure-activity relationship (SAR) efforts around our initial lead compound 1 led to the identification of potent P2X7 receptor antagonists with improved pharmacokinetic profiles. These compounds were potent and selective at the P2X7 receptor in both human and rodent. Compound (entry 31) exhibited oral efficacy in the rat MIA and CCI pain models. 相似文献
12.
The P2X7 purinoceptor is unique amongst the P2X receptor family in that its activation is able to stimulate the release of mature,
biologically active interleukin-1β (IL-1β), as well as a variety of other proinflammatory cytokines. Coupled with the predominate
localisation of this receptor to immunocytes of haemopoetic origin, this receptor is an obvious candidate to play a major
and pivotal role in processes of pain and inflammation. Using genetically modified animals that lack the P2X7 receptor, several investigators have shown that these mice do indeed demonstrate a blunted inflammatory response, and fail
to develop pain following both inflammatory and neuropathic insult. These animals also show altered cytokine production in
response to inflammatory stimulus, which is far broader than merely modulation of IL-1β release. In this short article, we
review the role of the P2X7 receptor in modulating the release of cytokines and other mediators, and discuss the findings made from P2X7 receptor-deficient animals. As well as highlighting outstanding questions regarding this intriguing receptor, we also speculate
as to the potential therapeutic benefit of P2X7 receptor modulation. 相似文献
13.
Miras-Portugal MT Díaz-Hernández M Giráldez L Hervás C Gómez-Villafuertes R Sen RP Gualix J Pintor J 《Neurochemical research》2003,28(10):1597-1605
ATP stimulates [Ca2+]i increases in midbrain synaptosomes via specific ionotropic receptors (P2X receptors). Previous studies have demonstrated the implication of P2X3 subunits in these responses, but additional P2X subunits must be involved. In the present study, ATP and BzATP proved to be able to induce intrasynaptosomal calcium transients in the midbrain synaptosomes, their effects being potentiated when assayed in a Mg2+-free medium. Indeed, BzATP was shown to be more potent than ATP, and their effects could be inhibited by PPADS and KN-62, but not by suramin. This activity profile is consistent with the presence of functional P2X7 receptors in the midbrain terminals. The existence of presynaptic responses to selective P2X7 agonists could be confirmed by means of a microfluorimetric technique allowing [Ca2+]i measurements in single synaptic terminals. Additionally, the P2X7 receptor protein could be identified in the midbrain synaptosomes and in axodendritic prolongations of cerebellar granule cells by immunochemical staining. 相似文献
14.
Lucia Corso Anna Cavallero Debora Baroni Patrizia Garbati Gianfranco Prestipino Silvia Bisti Mario Nobile Cristiana Picco 《Purinergic signalling》2016,12(1):161-174
P2X7-type purinergic receptors are distributed throughout the nervous system where they contribute to physiological and pathological functions. In the retina, this receptor is found in both inner and outer cells including microglia modulating signaling and health of retinal cells. It is involved in retinal neurodegenerative disorders such as retinitis pigmentosa and age-related macular degeneration (AMD). Experimental studies demonstrated that saffron protects photoreceptors from light-induced damage preserving both retinal morphology and visual function and improves retinal flicker sensitivity in AMD patients. To evaluate a possible interaction between saffron and P2X7 receptors (P2X7Rs), different cellular models and experimental approaches were used. We found that saffron positively influences the viability of mouse primary retinal cells and photoreceptor-derived 661W cells exposed to ATP, and reduced the ATP-induced intracellular calcium increase in 661W cells. Similar results were obtained on HEK cells transfected with recombinant rat P2X7R but not on cells transfected with rat P2X2R. Finally, patch-clamp experiments showed that saffron inhibited cationic currents in HEK-P2X7R cells. These results point out a novel mechanism through which saffron may exert its protective role in neurodegeneration and support the idea that P2X7-mediated calcium signaling may be a crucial therapeutic target in the treatment of neurodegenerative diseases. 相似文献
15.
Li X Zhang J Gao Y Yang Y Xu C Li G Guo G Liu S Xie J Liang S 《Purinergic signalling》2011,7(4):489-497
Pain is a major problem after burns. Procedural pain evoked by burn dressing changes is common in patients, and its management
is a critical part of treatment in acute burn injuries. Burn pain is very likely the most difficult form of acute pain to
treat. ATP contributes to inflammation, and ATP is implicated in peripheral pain signaling via actions upon P2X3 receptors. Puerarin is extracted from a traditional Chinese medicine and may act on P2X3 receptor mechanisms. The Visual Analogue Scale (VAS) has been shown to be a sensitive indicator of pain intensity and treatment
effects. Peripheral blood mononuclear cells (PBMCs) are involved in nociception or pain after burn injury. Burn patients were
randomly divided into normal saline (NS) group (salt solution is saline) and puerarin-treated group and pain (Visual Analogue
Scale scores) and inflammation (PBMCs) measured. Burn pain produces a stress response, so blood glucose, insulin, and cortisol
levels in burn patients were determined. Furthermore, the expression of P2X3 protein and mRNA in PBMCs was detected. The VAS scores in the puerarin-treated group were lower than those in NS group. The
blood glucose, insulin, and cortisol levels in the puerarin-treated group at post-dressing changes were significantly decreased
in comparison with those in NS group. The expression levels of P2X3 protein and mRNA in PBMCs of burn patients in NS group were significantly increased in comparison with those in the puerarin-treated
group. Puerarin can antagonize inflammatory factors (such as ATP) and decrease the upregulated expressions of P2X3 protein and mRNA in PBMCs after burns to decrease VAS. Thus, puerarin had an analgesic effect on procedural pain in dressing
changes of burn patients related to P2X3 receptors. 相似文献
16.
A region 2 kb upstream of exon 1 of the P2X7 gene was sequenced using DNA from nine healthy individuals who exhibited three different ATP response phenotypes (i.e. high, low and interferon gamma-inducible). Five single nucleotide polymorphisms were identified within the nine donor promoter sequences but none were associated with a specific ATP response phenotype. A P2X7 loss of function polymorphism (1513 in exon 13) was also screened for within donor DNA but no response associations were identified. ATP response phenotype was positively associated with P2X(7) receptor expression, as assessed by flow cytometry, but not with any identified receptor or promoter gene polymorphisms. 相似文献
17.
Vial C Fung CY Goodall AH Mahaut-Smith MP Evans RJ 《Biochemical and biophysical research communications》2006,343(2):415-419
ATP-stimulated P2X1 and ADP-stimulated P2Y1 receptors play important roles in platelet activation. An increase in intracellular Ca2+ represents a key signalling event coupled to both of these receptors, mediated via direct gating of Ca2+-permeable channels in the case of P2X1 and phospholipase-C-dependent Ca2+ mobilisation for P2Y1. We show that disruption of cholesterol-rich membrane lipid rafts reduces P2X1 receptor-mediated calcium increases by approximately 80%, while P2Y1 receptor-dependent Ca2+ release is unaffected. In contrast to artery, vas deferens, bladder smooth muscle, and recombinant expression in cell lines, where P2X1 receptors show almost exclusive association with lipid rafts, only approximately 20% of platelet P2X1 receptors are co-expressed with the lipid raft marker flotillin-2. We conclude that lipid rafts play a significant role in the regulation of P2X1 but not P2Y1 receptors in human platelets and that a reserve of non-functional P2X1 receptors may exist. 相似文献
18.
Brumfield S Matasi JJ Tulshian D Czarniecki M Greenlee W Garlisi C Qiu H Devito K Chen SC Sun Y Bertorelli R Ansell J Geiss W Le VD Martin GS Vellekoop SA Haber J Allard ML 《Bioorganic & medicinal chemistry letters》2011,21(24):7287-7290
Novel P2X7 antagonists were developed using a purine scaffold. These compounds were potent and selective at the P2X7 receptor in human and rodent as well as efficacious in rodent pain models. Compound 15a was identified to have oral potency in several pain models in rodent similar to naproxen, gabapentin and pregabalin. Structure–activity relationship (SAR) development and results of pain models are presented. 相似文献
19.
20.
Bradley HJ Liu X Collins V Owide J Goli GR Smith M Surprenant A White SJ Jiang LH 《FEBS letters》2010,584(23):4740-4744
We investigated membrane targeting of the P2X7 receptor (P2X7R) in polarized epithelial cells using immunofluorescent confocal imaging. The wild-type receptor was targeted to the basolateral membrane, independently of adaptor protein μ1B. Deletion of the majority of the intracellular C-terminus, or the last 26 residues (P570-Y595), conferred targeting of the protein to the apical membrane. Alanine substitution in the microdomain P582-Q587 caused similar apical membrane targeting without major effect on the receptor function and surface expression. Our results show basolateral membrane targeting of the P2X7R in epithelial cells and that the intracellular C-terminal microdomain P582-Q587 is crucial in this process.