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Sample records for p2x receptor channel

  1. Epithelial P2X purinergic receptor channel expression and function

    PubMed Central

    Taylor, Amanda L.; Schwiebert, Lisa M.; Smith, Jeffrey J.; King, Chris; Jones, Julie R.; Sorscher, Eric J.; Schwiebert, Erik M.

    1999-01-01

    P2X purinergic receptor (P2XR) channels bind ATP and mediate Ca2+ influx — 2 signals that stimulate secretory Cl– transport across epithelia. We tested the hypotheses that P2XR channels are expressed by epithelia and that P2XRs transduce extracellular ATP signals into stimulation of Cl– transport across epithelia. Electrophysiological data and mRNA analysis of human and mouse pulmonary epithelia and other epithelial cells indicate that multiple P2XRs are broadly expressed in these tissues and that they are active on both apical and basolateral surfaces. Because P2X-selective agonists bind multiple P2XR subtypes, and because P2X agonists stimulate Cl– transport across nasal mucosa of cystic fibrosis (CF) patients as well as across non-CF nasal mucosa, P2XRs may provide novel targets for extracellular nucleotide therapy of CF. PMID:10510328

  2. P2X receptors.

    PubMed

    North, R Alan

    2016-08-01

    Extracellular adenosine 5'-triphosphate (ATP) activates cell surface P2X and P2Y receptors. P2X receptors are membrane ion channels preferably permeable to sodium, potassium and calcium that open within milliseconds of the binding of ATP. In molecular architecture, they form a unique structural family. The receptor is a trimer, the binding of ATP between subunits causes them to flex together within the ectodomain and separate in the membrane-spanning region so as to open a central channel. P2X receptors have a widespread tissue distribution. On some smooth muscle cells, P2X receptors mediate the fast excitatory junction potential that leads to depolarization and contraction. In the central nervous system, activation of P2X receptors allows calcium to enter neurons and this can evoke slower neuromodulatory responses such as the trafficking of receptors for the neurotransmitter glutamate. In primary afferent nerves, P2X receptors are critical for the initiation of action potentials when they respond to ATP released from sensory cells such as taste buds, chemoreceptors or urothelium. In immune cells, activation of P2X receptors triggers the release of pro-inflammatory cytokines such as interleukin 1β. The development of selective blockers of different P2X receptors has led to clinical trials of their effectiveness in the management of cough, pain, inflammation and certain neurodegenerative diseases.This article is part of the themed issue 'Evolution brings Ca(2+) and ATP together to control life and death'. PMID:27377721

  3. Activation and Regulation of Purinergic P2X Receptor Channels

    PubMed Central

    Coddou, Claudio; Yan, Zonghe; Obsil, Tomas; Huidobro-Toro, J. Pablo

    2011-01-01

    Mammalian ATP-gated nonselective cation channels (P2XRs) can be composed of seven possible subunits, denoted P2X1 to P2X7. Each subunit contains a large ectodomain, two transmembrane domains, and intracellular N and C termini. Functional P2XRs are organized as homomeric and heteromeric trimers. This review focuses on the binding sites involved in the activation (orthosteric) and regulation (allosteric) of P2XRs. The ectodomains contain three ATP binding sites, presumably located between neighboring subunits and formed by highly conserved residues. The detection and coordination of three ATP phosphate residues by positively charged amino acids are likely to play a dominant role in determining agonist potency, whereas an AsnPheArg motif may contribute to binding by coordinating the adenine ring. Nonconserved ectodomain histidines provide the binding sites for trace metals, divalent cations, and protons. The transmembrane domains account not only for the formation of the channel pore but also for the binding of ivermectin (a specific P2X4R allosteric regulator) and alcohols. The N- and C- domains provide the structures that determine the kinetics of receptor desensitization and/or pore dilation and are critical for the regulation of receptor functions by intracellular messengers, kinases, reactive oxygen species and mercury. The recent publication of the crystal structure of the zebrafish P2X4.1R in a closed state provides a major advance in the understanding of this family of receptor channels. We will discuss data obtained from numerous site-directed mutagenesis experiments accumulated during the last 15 years with reference to the crystal structure, allowing a structural interpretation of the molecular basis of orthosteric and allosteric ligand actions. PMID:21737531

  4. Post-translational regulation of P2X receptor channels: modulation by phospholipids

    PubMed Central

    Bernier, Louis-Philippe; Ase, Ariel R.; Séguéla, Philippe

    2013-01-01

    P2X receptor channels mediate fast excitatory signaling by ATP and play major roles in sensory transduction, neuro-immune communication and inflammatory response. P2X receptors constitute a gene family of calcium-permeable ATP-gated cation channels therefore the regulation of P2X signaling is critical for both membrane potential and intracellular calcium homeostasis. Phosphoinositides (PIPn) are anionic signaling phospholipids that act as functional regulators of many types of ion channels. Direct PIPn binding was demonstrated for several ligand- or voltage-gated ion channels, however no generic motif emerged to accurately predict lipid-protein binding sites. This review presents what is currently known about the modulation of the different P2X subtypes by phospholipids and about critical determinants underlying their sensitivity to PIPn levels in the plasma membrane. All functional mammalian P2X subtypes tested, with the notable exception of P2X5, have been shown to be positively modulated by PIPn, i.e., homomeric P2X1, P2X2, P2X3, P2X4, and P2X7, as well as heteromeric P2X1/5 and P2X2/3 receptors. Based on various results reported on the aforementioned subtypes including mutagenesis of the prototypical PIPn-sensitive P2X4 and PIPn-insensitive P2X5 receptor subtypes, an increasing amount of functional, biochemical and structural evidence converges on the modulatory role of a short polybasic domain located in the proximal C-terminus of P2X subunits. This linear motif, semi-conserved in the P2X family, seems necessary and sufficient for encoding direct modulation of ATP-gated channels by PIPn. Furthermore, the physiological impact of the regulation of ionotropic purinergic responses by phospholipids on pain pathways was recently revealed in the context of native crosstalks between phospholipase C (PLC)-linked metabotropic receptors and P2X receptor channels in dorsal root ganglion sensory neurons and microglia. PMID:24324400

  5. Insights into the channel gating of P2X receptors from structures, dynamics and small molecules

    PubMed Central

    Wang, Jin; Yu, Ye

    2016-01-01

    P2X receptors, as ATP-gated non-selective trimeric ion channels, are permeable to Na+, K+ and Ca2+. Comparing with other ligand-gated ion channel families, P2X receptors are distinct in their unique gating properties and pathophysiological roles, and have attracted attention as promising drug targets for a variety of diseases, such as neuropathic pain, multiple sclerosis, rheumatoid arthritis and thrombus. Several small molecule inhibitors for distinct P2X subtypes have entered into clinical trials. However, many questions regarding the gating mechanism of P2X remain unsolved. The structural determinations of P2X receptors at the resting and ATP-bound open states revealed that P2X receptor gating is a cooperative allosteric process involving multiple domains, which marks the beginning of the post-structure era of P2X research at atomic level. Here, we review the current knowledge on the structure-function relationship of P2X receptors, depict the whole picture of allosteric changes during the channel gating, and summarize the active sites that may contribute to new strategies for developing novel allosteric drugs targeting P2X receptors. PMID:26725734

  6. Subtype-specific control of P2X receptor channel signaling by ATP and Mg2+

    PubMed Central

    Li, Mufeng; Silberberg, Shai D.; Swartz, Kenton J.

    2013-01-01

    The identity and forms of activating ligands for ion channels are fundamental to their physiological roles in rapid electrical signaling. P2X receptor channels are ATP-activated cation channels that serve important roles in sensory signaling and inflammation, yet the active forms of the nucleotide are unknown. In physiological solutions, ATP is ionized and primarily found in complex with Mg2+. Here we investigated the active forms of ATP and found that the action of MgATP2− and ATP4− differs between subtypes of P2X receptors. The slowly desensitizing P2X2 receptor can be activated by free ATP, but MgATP2− promotes opening with very low efficacy. In contrast, both free ATP and MgATP2− robustly open the rapidly desensitizing P2X3 subtype. A further distinction between these two subtypes is the ability of Mg2+ to regulate P2X3 through a distinct allosteric mechanism. Importantly, heteromeric P2X2/3 channels present in sensory neurons exhibit a hybrid phenotype, characterized by robust activation by MgATP2− and weak regulation by Mg2+. These results reveal the existence of two classes of homomeric P2X receptors with differential sensitivity to MgATP2− and regulation by Mg2+, and demonstrate that both restraining mechanisms can be disengaged in heteromeric channels to form fast and sensitive ATP signaling pathways in sensory neurons. PMID:23959888

  7. Structural Insights into Divalent Cation Modulations of ATP-Gated P2X Receptor Channels.

    PubMed

    Kasuya, Go; Fujiwara, Yuichiro; Takemoto, Mizuki; Dohmae, Naoshi; Nakada-Nakura, Yoshiko; Ishitani, Ryuichiro; Hattori, Motoyuki; Nureki, Osamu

    2016-02-01

    P2X receptors are trimeric ATP-gated cation channels involved in physiological processes ranging widely from neurotransmission to pain and taste signal transduction. The modulation of the channel gating, including that by divalent cations, contributes to these diverse physiological functions of P2X receptors. Here, we report the crystal structure of an invertebrate P2X receptor from the Gulf Coast tick Amblyomma maculatum in the presence of ATP and Zn(2+) ion, together with electrophysiological and computational analyses. The structure revealed two distinct metal binding sites, M1 and M2, in the extracellular region. The M1 site, located at the trimer interface, is responsible for Zn(2+) potentiation by facilitating the structural change of the extracellular domain for pore opening. In contrast, the M2 site, coupled with the ATP binding site, might contribute to regulation by Mg(2+). Overall, our work provides structural insights into the divalent cation modulations of P2X receptors. PMID:26804916

  8. Molecular mechanism of ATP binding and ion channel activation in P2X receptors

    SciTech Connect

    Hattori, Motoyuki; Gouaux, Eric

    2012-10-24

    P2X receptors are trimeric ATP-activated ion channels permeable to Na{sup +}, K{sup +} and Ca{sup 2+}. The seven P2X receptor subtypes are implicated in physiological processes that include modulation of synaptic transmission, contraction of smooth muscle, secretion of chemical transmitters and regulation of immune responses. Despite the importance of P2X receptors in cellular physiology, the three-dimensional composition of the ATP-binding site, the structural mechanism of ATP-dependent ion channel gating and the architecture of the open ion channel pore are unknown. Here we report the crystal structure of the zebrafish P2X4 receptor in complex with ATP and a new structure of the apo receptor. The agonist-bound structure reveals a previously unseen ATP-binding motif and an open ion channel pore. ATP binding induces cleft closure of the nucleotide-binding pocket, flexing of the lower body {beta}-sheet and a radial expansion of the extracellular vestibule. The structural widening of the extracellular vestibule is directly coupled to the opening of the ion channel pore by way of an iris-like expansion of the transmembrane helices. The structural delineation of the ATP-binding site and the ion channel pore, together with the conformational changes associated with ion channel gating, will stimulate development of new pharmacological agents.

  9. Dynamic aspects of functional regulation of the ATP receptor channel P2X2.

    PubMed

    Kubo, Yoshihiro; Fujiwara, Yuichiro; Keceli, Batu; Nakajo, Koichi

    2009-11-15

    The P2X(2) channel is a ligand-gated channel activated by ATP. Functional features that reflect the dynamic flexibility of the channel include time-dependent pore dilatation following ATP application and direct inhibitory interaction with activated nicotinic acetylcholine receptors on the membrane. We have been studying the mechanisms by which P2X(2) channel functionality is dynamically regulated. Using a Xenopus oocyte expression system, we observed that the pore properties, including ion selectivity and rectification, depend on the open channel density on the membrane. Pore dilatation was apparent when the open channel density was high and inward rectification was modest. We also observed that P2X(2) channels show voltage dependence, despite the absence of a canonical voltage sensor. At a semi-steady state after ATP application, P2X(2) channels were activated upon membrane hyperpolarization. This voltage-dependent activation was also [ATP] dependent. With increases in [ATP], the speed of hyperpolarization-induced activation was increased and the conductance-voltage relationship was shifted towards depolarized potentials. Based on analyses of experimental data and various simulations, we propose that these phenomena can be explained by assuming a fast ATP binding step and a rate-limiting voltage-dependent gating step. Complete elucidation of these regulatory mechanisms awaits dynamic imaging of functioning P2X(2) channels. PMID:19752115

  10. Pore properties and pharmacological features of the P2X receptor channel in airway ciliated cells

    PubMed Central

    Ma, Weiyuan; Korngreen, Alon; Weil, Simy; Cohen, Enbal Ben-Tal; Priel, Avi; Kuzin, Liubov; Silberberg, Shai D

    2006-01-01

    Airway ciliated cells express an ATP-gated P2X receptor channel of unknown subunit composition (P2Xcilia) which is modulated by Na+ and by long exposures to ATP. P2Xcilia was investigated by recording currents from freshly dissociated rabbit airway ciliated cells with the patch-clamp technique in the whole-cell configuration. During the initial continuous exposure to extracellular ATP, P2Xcilia currents gradually increase in magnitude (priming), yet the permeability to N-methyl-d-glucamine (NMDG) does not change, indicating that priming does not arise from a progressive change in pore diameter. Na+, which readily permeates P2Xcilia receptor channels, was found to inhibit the channel extracellular to the electric field. The rank order of permeability to various monovalent cations is: Li+, Na+, K+, Rb+, Cs+, NMDG+ and TEA+, with a relative permeability of 1.35, 1.0, 0.99, 0.91, 0.79, 0.19 and 0.10, respectively. The rank order for the alkali cations follows an Eisenman series XI for a high-strength field site. Ca2+ has been estimated to be 7-fold more permeant than Na+. The rise in [Ca2+]i in ciliated cells, induced by the activation of P2Xcilia, is largely inhibited by either Brilliant Blue G or KN-62, indicating that P2X7 may be a part of P2Xcilia. P2Xcilia is augmented by Zn2+ and by ivermectin, and P2X4 receptor protein is detected by immunolabelling at the basal half of the cilia, strongly suggesting that P2X4 is a component of P2Xcilia receptor channels. Taken together, these results suggest that P2Xcilia is either assembled from P2X4 and P2X7 subunits, or formed from modified P2X4 subunits. PMID:16423852

  11. Lipopolysaccharide Inhibits the Channel Activity of the P2X7 Receptor

    PubMed Central

    Leiva-Salcedo, Elias; Coddou, Claudio; Rodríguez, Felipe E.; Penna, Antonello; Lopez, Ximena; Neira, Tanya; Fernández, Ricardo; Imarai, Mónica; Rios, Miguel; Escobar, Jorge; Montoya, Margarita; Huidobro-Toro, J. Pablo; Escobar, Alejandro; Acuña-Castillo, Claudio

    2011-01-01

    The purinergic P2X7 receptor (P2X7R) plays an important role during the immune response, participating in several events such as cytokine release, apoptosis, and necrosis. The bacterial endotoxin lipopolysaccharide (LPS) is one of the strongest stimuli of the immune response, and it has been shown that P2X7R activation can modulate LPS-induced responses. Moreover, a C-terminal binding site for LPS has been proposed. In order to evaluate if LPS can directly modulate the activity of the P2X7R, we tested several signaling pathways associated with P2X7R activation in HEK293 cells that do not express the TLR-4 receptor. We found that LPS alone was unable to induce any P2X7R-related activity, suggesting that the P2X7R is not directly activated by the endotoxin. On the other hand, preapplication of LPS inhibited ATP-induced currents, intracellular calcium increase, and ethidium bromide uptake and had no effect on ERK activation in HEK293 cells. In splenocytes-derived T-regulatory cells, in which ATP-induced apoptosis is driven by the P2X7R, LPS inhibited ATP-induced apoptosis. Altogether, these results demonstrate that LPS modulates the activity of the P2X7R and suggest that this effect could be of physiological relevance. PMID:21941410

  12. Lipopolysaccharide inhibits the channel activity of the P2X7 receptor.

    PubMed

    Leiva-Salcedo, Elias; Coddou, Claudio; Rodríguez, Felipe E; Penna, Antonello; Lopez, Ximena; Neira, Tanya; Fernández, Ricardo; Imarai, Mónica; Rios, Miguel; Escobar, Jorge; Montoya, Margarita; Huidobro-Toro, J Pablo; Escobar, Alejandro; Acuña-Castillo, Claudio

    2011-01-01

    The purinergic P2X7 receptor (P2X7R) plays an important role during the immune response, participating in several events such as cytokine release, apoptosis, and necrosis. The bacterial endotoxin lipopolysaccharide (LPS) is one of the strongest stimuli of the immune response, and it has been shown that P2X7R activation can modulate LPS-induced responses. Moreover, a C-terminal binding site for LPS has been proposed. In order to evaluate if LPS can directly modulate the activity of the P2X7R, we tested several signaling pathways associated with P2X7R activation in HEK293 cells that do not express the TLR-4 receptor. We found that LPS alone was unable to induce any P2X7R-related activity, suggesting that the P2X7R is not directly activated by the endotoxin. On the other hand, preapplication of LPS inhibited ATP-induced currents, intracellular calcium increase, and ethidium bromide uptake and had no effect on ERK activation in HEK293 cells. In splenocytes-derived T-regulatory cells, in which ATP-induced apoptosis is driven by the P2X7R, LPS inhibited ATP-induced apoptosis. Altogether, these results demonstrate that LPS modulates the activity of the P2X7R and suggest that this effect could be of physiological relevance. PMID:21941410

  13. A new class of ligand-gated ion channel defined by P2x receptor for extracellular ATP.

    PubMed

    Valera, S; Hussy, N; Evans, R J; Adami, N; North, R A; Surprenant, A; Buell, G

    1994-10-01

    Extracellular ATP exerts its effects through P2 purinoceptors: these are ligand-gated ion channels (P2x) or G-protein-coupled receptors (P2Y, P2U). ATP at P2x receptors mediates synaptic transmission between neurons and from neurons to smooth muscle, being responsible, for example, for sympathetic vasoconstriction in small arteries and arterioles. We have now cloned a complementary DNA encoding the P2x receptor from rat vas deferens and expressed it in Xenopus oocytes and mammalian cells. ATP activates a cation-selective ion channel with relatively high calcium permeability. Structural predictions suggest that the protein (399 amino acids long) is mostly extracellular and contains only two transmembrane domains plus a pore-forming motif which resembles that of potassium channels. The P2x receptor thus defines a new family of ligand-gated ion channels. PMID:7523951

  14. G-protein-coupled receptor regulation of P2X1 receptors does not involve direct channel phosphorylation

    PubMed Central

    2004-01-01

    P2X1 receptors for ATP are ligand-gated cation channels, which mediate smooth muscle contraction, contribute to blood clotting and are co-expressed with a range of GPCRs (G-protein-coupled receptors). Stimulation of Gαq-coupled mGluR1α (metabotropic glutamate receptor 1α), P2Y1 or P2Y2 receptors co-expressed with P2X1 receptors in Xenopus oocytes evoked calcium-activated chloride currents (IClCa) and potentiated subsequent P2X1-receptor-mediated currents by up to 250%. The mGluR1α-receptor-mediated effects were blocked by the phospholipase C inhibitor U-73122. Potentiation was mimicked by treatment with the phor-bol ester PMA. P2X receptors have a conserved intracellular PKC (protein kinase C) site; however, GPCR- and PMA-mediated potentiation was still observed with point mutants in which this site was disrupted. Similarly, the potentiation by GPCRs or PMA was unaffected by chelating the intracellular calcium rise with BAPTA/AM [bis(o-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid tetrakis-(acetoxymethyl ester)] or the PKC inhibitors Ro-32-0432 and bisindolylmaleimide I, suggesting that the regulation does not involve a calcium-sensitive form of PKC. However, both GPCR and PMA potentiation were blocked by the kinase inhibitor staurosporine. Potentiation by phorbol esters was recorded in HEK-293 cells expressing P2X1 receptors, and radiolabelling of phosphorylated proteins in these cells demonstrated that P2X1 receptors are basally phosphorylated and that this level of phosphorylation is unaffected by phorbol ester treatment. This demonstrates that P2X1 regulation does not result directly from phosphorylation of the channel, but more likely by a staurosporine-sensitive phosphorylation of an accessory protein in the P2X1 receptor complex and suggests that in vivo fine-tuning of P2X1 receptors by GPCRs may contribute to cardiovascular control and haemostasis. PMID:15144237

  15. P2X Receptors as Drug Targets

    PubMed Central

    Jarvis, Michael F.

    2013-01-01

    The study of P2X receptors has long been handicapped by a poverty of small-molecule tools that serve as selective agonists and antagonists. There has been progress, particularly in the past 10 years, as cell-based high-throughput screening methods were applied, together with large chemical libraries. This has delivered some drug-like molecules in several chemical classes that selectively target P2X1, P2X3, or P2X7 receptors. Some of these are, or have been, in clinical trials for rheumatoid arthritis, pain, and cough. Current preclinical research programs are studying P2X receptor involvement in pain, inflammation, osteoporosis, multiple sclerosis, spinal cord injury, and bladder dysfunction. The determination of the atomic structure of P2X receptors in closed and open (ATP-bound) states by X-ray crystallography is now allowing new approaches by molecular modeling. This is supported by a large body of previous work using mutagenesis and functional expression, and is now being supplemented by molecular dynamic simulations and in silico ligand docking. These approaches should lead to P2X receptors soon taking their place alongside other ion channel proteins as therapeutically important drug targets. PMID:23253448

  16. Metabotropic P2Y receptors inhibit P2X3 receptor-channels via G protein-dependent facilitation of their desensitization

    PubMed Central

    Gerevich, Z; Zadori, Z; Müller, C; Wirkner, K; Schröder, W; Rubini, P; Illes, P

    2007-01-01

    Background and purpose: The aim of the present study was to investigate whether the endogenous metabotropic P2Y receptors modulate ionotropic P2X3 receptor-channels. Experimental approach: Whole-cell patch-clamp experiments were carried out on HEK293 cells permanently transfected with human P2X3 receptors (HEK293-hP2X3 cells) and rat dorsal root ganglion (DRG) neurons. Key results: In both cell types, the P2Y1,12,13 receptor agonist, ADP-β-S, inhibited P2X3 currents evoked by the selective agonist, α,β-methylene ATP (α,β-meATP). This inhibition could be markedly counteracted by replacing in the pipette solution the usual GTP with GDP-β-S, a procedure known to block all G protein heterotrimers. P2X3 currents evoked by ATP, activating both P2Y and P2X receptors, caused a smaller peak amplitude and desensitized faster than those currents evoked by the selective P2X3 receptor agonist α,β-meATP. In the presence of intracellular GDP-β-S, ATP- and α,β-meATP-induced currents were identical. Recovery from P2X3 receptor desensitization induced by repetitive ATP application was slower than the recovery from α,β-meATP-induced desensitization. When G proteins were blocked by intracellular GDP-β-S, the recovery from the ATP- and α,β-meATP-induced desensitization were of comparable speed. Conclusions and Implications: Our results suggest that the activation of P2Y receptors G protein-dependently facilitates the desensitization of P2X3 receptors and suppresses the recovery from the desensitized state. Hence, the concomitant stimulation of P2X3 and P2Y receptors of DRG neurons by ATP may result both in an algesic effect and a partly counterbalancing analgesic activity. PMID:17351651

  17. Calcium permeability and block at homomeric and heteromeric P2X2 and P2X3 receptors, and P2X receptors in rat nodose neurones

    PubMed Central

    Virginio, Caterina; North, R A; Surprenant, Annmarie

    1998-01-01

    Whole-cell recordings were made from HEK 293 (human embryonic kidney) cells stably transfected with cDNAs encoding P2X2, P2X3 or both receptors (P2X2/3) and from cultured rat nodose neurones. Nodose neurones all showed immunoreactivity for both P2X2 and P2X3, but not P2X1, receptors. Reversal potentials were measured in extracellular sodium, N-methyl-D-glucamine (NMDG) and NMDG containing 5 mM Ca2+; the values were used to compute relative permeabilities (PNMDG/PNa and PCa/PNa). PNMDG/PNa was not different for P2X2, P2X2/3 and nodose neurones (0.03) but was significantly higher (0.07) for P2X3 receptors. PCa/PNa was not different among P2X3, P2X2/3 and nodose neurones (1.2-1.5) but was significantly higher (2.5) for P2X2 receptors. External Ca2+ inhibited purinoceptor currents with half-maximal concentrations of 5 mM at the P2X2 receptor, 89 mM at the P2X3 receptor and 15 mM at both the P2X2/3 heteromeric receptor and nodose neurones. In each case, the inhibition was voltage independent and was overcome by increasing concentrations of agonist. These results may indicate that Ca2+ permeability of the heteromeric (P2X2/3) channel is dominated by that of the P2X3 subunit, while Ca2+ block of the receptor involves both P2X2 and P2X3 subunits. The correspondence in properties between P2X2/3 receptors and nodose ganglion neurones further supports the conclusion that the native α,β-methylene ATP-sensitive receptor is a P2X2/3 heteromultimer. PMID:9625864

  18. Structural and Molecular Modeling Features of P2X Receptors

    PubMed Central

    Alves, Luiz Anastacio; da Silva, João Herminio Martins; Ferreira, Dinarte Neto Moreira; Fidalgo-Neto, Antonio Augusto; Teixeira, Pedro Celso Nogueira; de Souza, Cristina Alves Magalhães; Caffarena, Ernesto Raúl; de Freitas, Mônica Santos

    2014-01-01

    Currently, adenosine 5′-triphosphate (ATP) is recognized as the extracellular messenger that acts through P2 receptors. P2 receptors are divided into two subtypes: P2Y metabotropic receptors and P2X ionotropic receptors, both of which are found in virtually all mammalian cell types studied. Due to the difficulty in studying membrane protein structures by X-ray crystallography or NMR techniques, there is little information about these structures available in the literature. Two structures of the P2X4 receptor in truncated form have been solved by crystallography. Molecular modeling has proven to be an excellent tool for studying ionotropic receptors. Recently, modeling studies carried out on P2X receptors have advanced our knowledge of the P2X receptor structure-function relationships. This review presents a brief history of ion channel structural studies and shows how modeling approaches can be used to address relevant questions about P2X receptors. PMID:24637936

  19. Ectodomain movements of an ATP-gated ion channel (P2X2 receptor) probed by disulfide locking.

    PubMed

    Stelmashenko, Olga; Compan, Vincent; Browne, Liam E; North, R Alan

    2014-04-01

    The ectodomain of the P2X receptor is formed mainly from two- or three-stranded β-sheets provided symmetrically by each of the three subunits. These enclose a central cavity that is closed off furthest from the plasma membrane (the turret) and that joins with the transmembrane helices to form the ion permeation pathway. Comparison of closed and open crystal structures indicates that ATP binds in a pocket positioned between strands provided by different subunits and that this flexes the β-sheets of the lower body and enlarges the central cavity: this pulls apart the outer ends of the transmembrane helices and thereby opens an aperture, or gate, where they intersect within the membrane bilayer. In the present work, we examined this opening model by introducing pairs of cysteines into the rat P2X2 receptor that might form disulfide bonds within or between subunits. Receptors were expressed in human embryonic kidney cells, and disulfide formation was assessed by observing the effect of dithiothreitol on currents evoked by ATP. Substitutions in the turret (P90C, P89C/S97C), body wall (S65C/S190C, S65C/D315C) and the transmembrane domains (V48C/I328C, V51C/I328C, S54C/I328C) strongly inhibited ATP-evoked currents prior to reduction with dithiothreitol. Western blotting showed that these channels also formed predominately as dimers and/or trimers rather than monomers. The results strongly support the channel opening mechanism proposed on the basis of available crystal structures. PMID:24515105

  20. P2X receptors: New players in cancer pain

    PubMed Central

    Franceschini, Alessia; Adinolfi, Elena

    2014-01-01

    Pain is unfortunately a quite common symptom for cancer patients. Normally pain starts as an episodic experience at early cancer phases to become chronic in later stages. In order to improve the quality of life of oncological patients, anti-cancer treatments are often accompanied by analgesic therapies. The P2X receptor are adenosine triphosphate (ATP) gated ion channels expressed by several cells including neurons, cancer and immune cells. Purinergic signaling through P2X receptors recently emerged as possible common pathway for cancer onset/growth and pain sensitivity. Indeed, tumor microenvironment is rich in extracellular ATP, which has a role in both tumor development and pain sensation. The study of the different mechanisms by which P2X receptors favor cancer progression and relative pain, represents an interesting challenge to design integrated therapeutic strategies for oncological patients. This review summarizes recent findings linking P2X receptors and ATP to cancer growth, progression and related pain. Special attention has been paid to the role of P2X2, P2X3, P2X4 and P2X7 in the genesis of cancer pain and to the function of P2X7 in tumor growth and metastasis. Therapeutic implications of the administration of different P2X receptor blockers to alleviate cancer-associated pain sensations contemporarily reducing tumor progression are also discussed. PMID:25426266

  1. P2X7 Receptors in Neurological and Cardiovascular Disorders

    PubMed Central

    Skaper, Stephen D.; Debetto, Patrizia; Giusti, Pietro

    2009-01-01

    P2X receptors are ATP-gated cation channels that mediate fast excitatory transmission in diverse regions of the brain and spinal cord. Several P2X receptor subtypes, including P2X7, have the unusual property of changing their ion selectivity during prolonged exposure to ATP, which results in a channel pore permeable to molecules as large as 900 daltons. The P2X7 receptor was originally described in cells of hematopoietic origin, and mediates the influx of Ca2+ and Na+ and Ca2+ and Na+ ions as well as the release of proinflammatory cytokines. P2X7 receptors may affect neuronal cell death through their ability to regulate the processing and release of interleukin-1β, a key mediator in neurodegeneration, chronic inflammation, and chronic pain. Activation of P2X7, a key mediator in neurodegeneration, chronic inflammation, and chronic pain. Activation of P2X7 receptors provides an inflammatory stimulus, and P2X7 receptor-deficient mice have substantially attenuated inflammatory responses, including models of neuropathic and chronic inflammatory pain. Moreover, P2X7 receptor activity, by regulating the release of proinflammatory cytokines, may be involved in the pathophysiology of depression. Apoptotic cell death occurs in a number of vascular diseases, including atherosclerosis, restenosis, and hypertension, and may be linked to the release of ATP from endothelial cells, P2X7 receptor activation, proinflammatory cytokine production, and endothelial cell apoptosis. In this context, the P2X7 receptor may be viewed as a gateway of communication between the nervous, immune, and cardiovascular systems. PMID:20029634

  2. Molecular characterization of purinergic receptor P2X4 involved in Japanese flounder (Paralichthys olivaceus) innate immune response and its interaction with ATP release channel Pannexin1.

    PubMed

    Li, Shuo; Chen, Xiaoli; Li, Xuejing; Geng, Xuyun; Lin, Rongxin; Li, Ming; Sun, Jinsheng

    2015-11-01

    P2X4 receptor (P2X4R) is a member of trimeric ATP-gated receptor channel family. Despite the importance of P2X4R in innate immunity has been addressed in mammals, the immunological significance of P2X4R has not been characterized in fish. In the present study we identified a full-length P2X4R cDNA sequence from Japanese flounder Paralichthys olivaceus (termed poP2X4R) by RT-PCR and RACE approaches and analyzed its gene expression patterns under normal and immune challenge conditions. Qualitative RT-PCR analyses revealed that poP2X4R has a widespread distribution in all examined tissues but dominantly expressed in hepatopancreas. In Japanese flounder head kidney macrophages and peripheral blood lymphocytes, poP2X4R was rapidly and significantly up-regulated by the immune challenges of LPS, poly(I:C) and zymosan. In addition, poP2X4R was up-regulated in spleen, head kidney and gill tissues by Edwardsiella tarda infections. Furthermore, we showed that poP2X4R is a membrane glycoprotein which could interact with ATP release channel Pannexin1, an important component in extracellular ATP-activated purinergic signaling pathways involved in Japanese flounder innate immune response. From a comparative immunological point of view, our results have provided new evidence for the involvement of extracellular ATP-gated P2XRs in fish innate immunity. PMID:26321132

  3. Imaging P2X4 receptor subcellular distribution, trafficking, and regulation using P2X4-pHluorin.

    PubMed

    Xu, Ji; Chai, Hua; Ehinger, Konstantin; Egan, Terrance M; Srinivasan, Rahul; Frick, Manfred; Khakh, Baljit S

    2014-07-01

    P2X4 receptors are adenosine triphosphate (ATP)-gated cation channels present on the plasma membrane (PM) and also within intracellular compartments such as vesicles, vacuoles, lamellar bodies (LBs), and lysosomes. P2X4 receptors in microglia are up-regulated in epilepsy and in neuropathic pain; that is to say, their total and/or PM expression levels increase. However, the mechanisms underlying up-regulation of microglial P2X4 receptors remain unclear, in part because it has not been possible to image P2X4 receptor distribution within, or trafficking between, cellular compartments. Here, we report the generation of pH-sensitive fluorescently tagged P2X4 receptors that permit evaluations of cell surface and total receptor pools. Capitalizing on information gained from zebrafish P2X4.1 crystal structures, we designed a series of mouse P2X4 constructs in which a pH-sensitive green fluorescent protein, superecliptic pHluorin (pHluorin), was inserted into nonconserved regions located within flexible loops of the P2X4 receptor extracellular domain. One of these constructs, in which pHluorin was inserted after lysine 122 (P2X4-pHluorin123), functioned like wild-type P2X4 in terms of its peak ATP-evoked responses, macroscopic kinetics, calcium flux, current-voltage relationship, and sensitivity to ATP. P2X4-pHluorin123 also showed pH-dependent fluorescence changes, and was robustly expressed on the membrane and within intracellular compartments. P2X4-pHluorin123 identified cell surface and intracellular fractions of receptors in HEK-293 cells, hippocampal neurons, C8-B4 microglia, and alveolar type II (ATII) cells. Furthermore, it showed that the subcellular fractions of P2X4-pHluorin123 receptors were cell and compartment specific, for example, being larger in hippocampal neuron somata than in C8-B4 cell somata, and larger in C8-B4 microglial processes than in their somata. In ATII cells, P2X4-pHluorin123 showed that P2X4 receptors were secreted onto the PM when LBs

  4. Imaging P2X4 receptor subcellular distribution, trafficking, and regulation using P2X4-pHluorin

    PubMed Central

    Xu, Ji; Chai, Hua; Ehinger, Konstantin; Egan, Terrance M.; Srinivasan, Rahul; Frick, Manfred

    2014-01-01

    P2X4 receptors are adenosine triphosphate (ATP)-gated cation channels present on the plasma membrane (PM) and also within intracellular compartments such as vesicles, vacuoles, lamellar bodies (LBs), and lysosomes. P2X4 receptors in microglia are up-regulated in epilepsy and in neuropathic pain; that is to say, their total and/or PM expression levels increase. However, the mechanisms underlying up-regulation of microglial P2X4 receptors remain unclear, in part because it has not been possible to image P2X4 receptor distribution within, or trafficking between, cellular compartments. Here, we report the generation of pH-sensitive fluorescently tagged P2X4 receptors that permit evaluations of cell surface and total receptor pools. Capitalizing on information gained from zebrafish P2X4.1 crystal structures, we designed a series of mouse P2X4 constructs in which a pH-sensitive green fluorescent protein, superecliptic pHluorin (pHluorin), was inserted into nonconserved regions located within flexible loops of the P2X4 receptor extracellular domain. One of these constructs, in which pHluorin was inserted after lysine 122 (P2X4-pHluorin123), functioned like wild-type P2X4 in terms of its peak ATP-evoked responses, macroscopic kinetics, calcium flux, current–voltage relationship, and sensitivity to ATP. P2X4-pHluorin123 also showed pH-dependent fluorescence changes, and was robustly expressed on the membrane and within intracellular compartments. P2X4-pHluorin123 identified cell surface and intracellular fractions of receptors in HEK-293 cells, hippocampal neurons, C8-B4 microglia, and alveolar type II (ATII) cells. Furthermore, it showed that the subcellular fractions of P2X4-pHluorin123 receptors were cell and compartment specific, for example, being larger in hippocampal neuron somata than in C8-B4 cell somata, and larger in C8-B4 microglial processes than in their somata. In ATII cells, P2X4-pHluorin123 showed that P2X4 receptors were secreted onto the PM when LBs

  5. ATP P2X3 receptors and neuronal sensitization

    PubMed Central

    Fabbretti, Elsa

    2013-01-01

    Increasing evidence indicates the importance of extracellular adenosine triphosphate (ATP) in the modulation of neuronal function. In particular, fine control of ATP release and the selective and discrete ATP receptor operation are crucial elements of the crosstalk between neuronal and non-neuronal cells in the peripheral and central nervous systems. In peripheral neurons, ATP signaling gives an important contribution to neuronal sensitization, especially that involved in neuropathic pain. Among other subtypes, P2X3 receptors expressed on sensory neurons are sensitive even to nanomolar concentrations of extracellular ATP, and therefore are important transducers of pain stimuli. P2X3 receptor function is highly sensitive to soluble factors like neuropeptides and neurotrophins, and is controlled by transduction mechanisms, protein-protein interactions and discrete membrane compartmentalization. More recent findings have demonstrated that P2X3 receptors interact with the synaptic scaffold protein calcium/calmodulin-dependent serine protein kinase (CASK) in a state dependent fashion, indicating that CASK plays a crucial role in the modulation of P2X3 receptor stability and efficiency. Activation of P2X3 receptors within CASK/P2X3 complex has important consequences for neuronal plasticity and possibly for the release of neuromodulators and neurotransmitters. Better understanding of the interactome machinery of P2X3 receptors and their integration with other receptors and channels on neuronal surface membranes, is proposed to be essential to unveil the process of neuronal sensitization and related, abnormal pain signaling. PMID:24363643

  6. P2X3 receptors and peripheral pain mechanisms

    PubMed Central

    North, R Alan

    2004-01-01

    ATP released from damaged or inflamed tissues can act at P2X receptors expressed on primary afferent neurones. The resulting depolarization can initiate action potentials that are interpreted centrally as pain. P2X3 subunits are found in a subset of small-diameter, primary afferent neurones, some of which are also sensitive to capsaicin. They can form homo-oligomeric channels, or they can assemble with P2X2 subunits into hetero-oligomers. Studies with antagonists selective for P2X3-containing receptors, experiments with antisense oligonucleotides to reduce P2X3 subunit levels, and behavioural testing of P2X3 knock-out mice, all suggest a role for the P2X2/3 receptor in the signalling of chronic inflammatory pain and some features of neuropathic pain. The availability of such tools and experimental approaches promises to accelerate our understanding of the other physiological roles for P2X receptors on primary afferent neurones. PMID:12832496

  7. AF-353, a novel, potent and orally bioavailable P2X3/P2X2/3 receptor antagonist

    PubMed Central

    Gever, Joel R; Soto, Rothschild; Henningsen, Robert A; Martin, Renee S; Hackos, David H; Panicker, Sandip; Rubas, Werner; Oglesby, Ian B; Dillon, Michael P; Milla, Marcos E; Burnstock, Geoffrey; Ford, Anthony PDW

    2010-01-01

    Background and purpose: Purinoceptors containing the P2X3 subunit (P2X3 homotrimeric and P2X2/3 heterotrimeric) are members of the P2X family of ion channels gated by ATP and may participate in primary afferent sensitization in a variety of pain-related diseases. The current work describes the in vitro pharmacological characteristics of AF-353, a novel, orally bioavailable, highly potent and selective P2X3/P2X2/3 receptor antagonist. Experimental approach: The antagonistic potencies (pIC50) of AF-353 for rat and human P2X3 and human P2X2/3 receptors were determined using methods of radioligand binding, intracellular calcium flux and whole cell voltage-clamp electrophysiology. Key results: The pIC50 estimates for these receptors ranged from 7.3 to 8.5, while concentrations 300-fold higher had little or no effect on other P2X channels or on an assortment of receptors, enzymes and transporter proteins. In contrast to A-317491 and TNP-ATP, competition binding and intracellular calcium flux experiments suggested that AF-353 inhibits activation by ATP in a non-competitive fashion. Favourable pharmacokinetic parameters were observed in rat, with good oral bioavailability (%F = 32.9), reasonable half-life (t1/2 = 1.63 h) and plasma-free fraction (98.2% protein bound). Conclusions and implications: The combination of a favourable pharmacokinetic profile with the antagonist potency and selectivity for P2X3 and P2X2/3 receptors suggests that AF-353 is an excellent in vivo tool compound for study of these channels in animal models and demonstrates the feasibility of identifying and optimizing molecules into potential clinical candidates, and, ultimately, into a novel class of therapeutics for the treatment of pain-related disorders. PMID:20590629

  8. Functional characterization of intracellular Dictyostelium discoideum P2X receptors.

    PubMed

    Ludlow, Melanie J; Durai, Latha; Ennion, Steven J

    2009-12-11

    Indicative of cell surface P2X ion channel activation, extracellular ATP evokes a rapid and transient calcium influx in the model eukaryote Dictyostelium discoideum. Five P2X-like proteins (dP2XA-E) are present in this organism. However, their roles in purinergic signaling are unclear, because dP2XA proved to have an intracellular localization on the contractile vacuole where it is thought to be required for osmoregulation. To determine functional properties of the remaining four dP2X-like proteins and to assess their cellular roles, we recorded membrane currents from expressed cloned receptors and generated a quintuple knock-out Dictyostelium strain devoid of dP2X receptors. ATP evoked inward currents at dP2XB and dP2XE receptors but not at dP2XC or dP2XD. beta,gamma-Imido-ATP was more potent than ATP at dP2XB but a weak partial agonist at dP2XE. Currents in dP2XB and dP2XE were strongly inhibited by Na(+) but insensitive to copper and the P2 receptor antagonists pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid and suramin. Unusual for P2X channels, dP2XA and dP2XB were also Cl(-)-permeable. The extracellular purinergic response to ATP persisted in p2xA/B/C/D/E quintuple knock-out Dictyostelium demonstrating that dP2X channels are not responsible. dP2XB, -C, -D, and -E were found to be intracellularly localized to the contractile vacuole with the ligand binding domain facing the lumen. However, quintuple p2xA/B/C/D/E null cells were still capable of regulating cell volume in water demonstrating that, contrary to previous findings, dP2X receptors are not required for osmoregulation. Responses to the calmodulin antagonist calmidazolium, however, were reduced in p2xA/B/C/D/E null cells suggesting that dP2X receptors play a role in intracellular calcium signaling. PMID:19833731

  9. Manipulation of P2X Receptor Activities by Light Stimulation

    PubMed Central

    Kim, Sang Seong

    2016-01-01

    P2X receptors are involved in amplification of inflammatory responses in peripheral nociceptive fibers and in mediating pain-related signals to the CNS. Control of P2X activation has significant importance in managing unwanted hypersensitive neuron responses. To overcome the limitations of chemical ligand treatment, optical stimulation methods of optogenetics and photoswitching achieve efficient control of P2X activation while allowing specificity at the target site and convenient stimulation by light illumination. There are many potential applications for photosensitive elements, such as improved uncaging methods, photoisomerizable ligands, photoswitches, and gold nanoparticles. Each technique has both advantages and downsides, and techniques are selected according to the purpose of the application. Technical advances not only provide novel approaches to manage inflammation or pain mediated by P2X receptors but also suggest a similar approach for controlling other ion channels. PMID:26884649

  10. Conformational flexibility of the agonist binding jaw of the human P2X3 receptor is a prerequisite for channel opening

    PubMed Central

    Kowalski, M; Hausmann, R; Dopychai, A; Grohmann, M; Franke, H; Nieber, K; Schmalzing, G; Illes, P; Riedel, T

    2014-01-01

    Background and Purpose It is assumed that ATP induces closure of the binding jaw of ligand-gated P2X receptors, which eventually results in the opening of the membrane channel and the flux of cations. Immobilization by cysteine mutagenesis of the binding jaw inhibited ATP-induced current responses, but did not allow discrimination between disturbances of binding, gating, subunit assembly or trafficking to the plasma membrane. Experimental Approach A molecular model of the pain-relevant human (h)P2X3 receptor was used to identify amino acid pairs, which were located at the lips of the binding jaw and did not participate in agonist binding but strongly approached each other even in the absence of ATP. Key Results A series of cysteine double mutant hP2X3 receptors, expressed in HEK293 cells or Xenopus laevis oocytes, exhibited depressed current responses to α,β-methylene ATP (α,β-meATP) due to the formation of spontaneous inter-subunit disulfide bonds. Reducing these bonds with dithiothreitol reversed the blockade of the α,β-meATP transmembrane current. Amino-reactive fluorescence labelling of the His-tagged hP2X3 receptor and its mutants expressed in HEK293 or X. laevis oocytes demonstrated the formation of inter-subunit cross links in cysteine double mutants and, in addition, confirmed their correct trimeric assembly and cell surface expression. Conclusions and Implications In conclusion, spontaneous tightening of the binding jaw of the hP2X3 receptor by inter-subunit cross-linking of cysteine residues substituted at positions not directly involved in agonist binding inhibited agonist-evoked currents without interfering with binding, subunit assembly or trafficking. PMID:24989924

  11. Modulation of P2X3 and P2X2/3 Receptors by Monoclonal Antibodies.

    PubMed

    Shcherbatko, Anatoly; Foletti, Davide; Poulsen, Kris; Strop, Pavel; Zhu, Guoyun; Hasa-Moreno, Adela; Melton Witt, Jody; Loo, Carole; Krimm, Stellanie; Pios, Ariel; Yu, Jessica; Brown, Colleen; Lee, John K; Stroud, Robert; Rajpal, Arvind; Shelton, David

    2016-06-01

    Purinergic homomeric P2X3 and heteromeric P2X2/3 receptors are ligand-gated cation channels activated by ATP. Both receptors are predominantly expressed in nociceptive sensory neurons, and an increase in extracellular ATP concentration under pathological conditions, such as tissue damage or visceral distension, induces channel opening, membrane depolarization, and initiation of pain signaling. Hence, these receptors are considered important therapeutic targets for pain management, and development of selective antagonists is currently progressing. To advance the search for novel analgesics, we have generated a panel of monoclonal antibodies directed against human P2X3 (hP2X3). We have found that these antibodies produce distinct functional effects, depending on the homomeric or heteromeric composition of the target, its kinetic state, and the duration of antibody exposure. The most potent antibody, 12D4, showed an estimated IC50 of 16 nm on hP2X3 after short term exposure (up to 18 min), binding to the inactivated state of the channel to inhibit activity. By contrast, with the same short term application, 12D4 potentiated the slow inactivating current mediated by the heteromeric hP2X2/3 channel. Extending the duration of exposure to ∼20 h resulted in a profound inhibition of both homomeric hP2X3 and heteromeric hP2X2/3 receptors, an effect mediated by efficient antibody-induced internalization of the channel from the plasma membrane. The therapeutic potential of mAb12D4 was assessed in the formalin, complete Freund's adjuvant, and visceral pain models. The efficacy of 12D4 in the visceral hypersensitivity model indicates that antibodies against P2X3 may have therapeutic potential in visceral pain indications. PMID:27129281

  12. P2X7 receptor at the heart of disease

    PubMed Central

    Vasileiou, Ei; Montero, R M; Turner, C M; Vergoulas, G

    2010-01-01

    Purinergic signaling is a crucial component of disease whose pathophysiological basis is now well established. This review focuses on P2X7, a unique bifunctional purinoreceptor that either opens a non selective cation channel or forms a large, cytolytic pore depending on agonist application and leading to membrane blebbing and to cell death either by necrosis or apoptosis. Activation of P2X7 receptor has been shown to stimulate the release of multiple proinflammatory cytokines by activated macrophages, with the IL-1b to be the most extensively studied among them. These findings were verified by the use of knockout P2X7 (-/-) mice. Update information coming from all fields of research implicate this receptor at the very heart of diseases such as rheumatoid arthritis, multiple sclerosis, depression, Alzheimer disease, and to kidney damage, in renal fibrosis and experimental nephritis. Clinical studies are currently underway with the newly developed selective antagonists for P2X7 receptor, the results of which are eagerly anticipated. These studies together with data from in-vivo experiments with the P2X7 knockout mice and in-vitro experiments will shed light in this exciting area. PMID:20981163

  13. Imaging P2X4 Receptor Lateral Mobility in Microglia

    PubMed Central

    Toulme, Estelle; Khakh, Baljit S.

    2012-01-01

    ATP-gated ionotropic P2X4 receptors are up-regulated in activated microglia and are critical for the development of neuropathic pain, a microglia-associated disorder. However, the nature of how plasma membrane P2X4 receptors are regulated in microglia is not fully understood. We used single-molecule imaging to track quantum dot-labeled P2X4 receptors to explore P2X4 receptor mobility in the processes of resting and activated microglia. We find that plasma membrane P2X4 receptor lateral mobility in resting microglial processes is largely random, consisting of mobile and slowly mobile receptors. Moreover, lateral mobility is P2X subunit- and cell-specific, increased in an ATP activation and calcium-dependent manner, and enhanced in activated microglia by the p38 MAPK pathway that selectively regulates slowly mobile receptors. Thus, our data indicate that P2X4 receptors are dynamically regulated mobile ATP sensors, sampling more of the plasma membrane in response to ATP and during the activated state of microglia that is associated with nervous system dysfunction. PMID:22393055

  14. Enteric P2X receptors as potential targets for drug treatment of the irritable bowel syndrome

    PubMed Central

    Galligan, James J

    2004-01-01

    The irritable bowel syndrome (IBS) is a gastrointestinal motility disorder affecting millions of patients. IBS symptoms include diarrhea, constipation and pain. The etiology of IBS is due partly to changes in the function of nerves supplying the gastrointestinal tract, immune system activation and to psychological factors. P2X receptors are multimeric ATP-gated cation channels expressed by neuronal and non-neuronal cells. Sensory nerve endings in the gastrointestinal tract express P2X receptors. ATP released from gastrointestinal cells activates P2X receptors on sensory nerve endings to stimulate motor reflexes and to transmit nociceptive signals. Antagonists acting at P2X receptors on sensory nerves could attenuate abdominal pain in IBS patients. Primary afferent neurons intrinsic to the gut, and enteric motor- and interneurons express P2X receptors. These neurons participate in motor reflexes. Agonists acting at enteric P2X receptors may enhance gastrointestinal propulsion and secretion, and these drugs could be useful for treating constipation-predominant IBS. Antagonists acting at enteric P2X receptors would decrease propulsion and secretion and they might be useful for treating diarrhea-predominant IBS. Current knowledge of P2X receptor distribution and function in the gut of laboratory animals provides a rational basis for further exploration of the therapeutic potential for drugs acting at P2X receptors in IBS patients. However, more information about P2X receptor distribution and function in the human gastrointestinal tract is needed. Data on the distribution and function of P2X receptors on gastrointestinal immune cells would also provide insights into the therapeutic potential of P2X receptor agents in IBS. PMID:15051631

  15. A mechanism of intracellular P2X receptor activation.

    PubMed

    Sivaramakrishnan, Venketesh; Fountain, Samuel J

    2012-08-17

    P2X receptors (P2XRs) are ATP-activated calcium-permeable ligand-gated ion channels traditionally viewed as sensors of extracellular ATP during diverse physiological processes including pain, inflammation, and taste. However, in addition to a cell surface residency P2XRs also populate the membranes of intracellular compartments, including mammalian lysosomes, phagosomes, and the contractile vacuole (CV) of the amoeba Dictyostelium. The function of intracellular P2XRs is unclear and represents a major gap in our understanding of ATP signaling. Here, we exploit the genetic versatility of Dictyostelium to investigate the effects of physiological concentrations of ATP on calcium signaling in isolated CVs. Within the CV, an acidic calcium store, P2XRs are orientated to sense luminal ATP. Application of ATP to isolated vacuoles leads to luminal translocation of ATP and release of calcium. Mechanisms of luminal ATP translocation and ATP-evoked calcium release share common pharmacology, suggesting that they are linked processes. The ability of ATP to mobilize stored calcium is reduced in vacuoles isolated from P2X(A)R knock-out amoeba and ablated in cells devoid of P2XRs. Pharmacological inhibition of luminal ATP translocation or depletion of CV calcium attenuates CV function in vivo, manifesting as a loss of regulatory cell volume decrease following osmotic swelling. We propose that intracellular P2XRs regulate vacuole activity by acting as calcium release channels, activated by translocation of ATP into the vacuole lumen. PMID:22736763

  16. Signal transmission within the P2X2 trimeric receptor.

    PubMed

    Keceli, Batu; Kubo, Yoshihiro

    2014-06-01

    P2X2 receptor channel, a homotrimer activated by the binding of extracellular adenosine triphosphate (ATP) to three intersubunit ATP-binding sites (each located ∼50 Å from the ion permeation pore), also shows voltage-dependent activation upon hyperpolarization. Here, we used tandem trimeric constructs (TTCs) harboring critical mutations at the ATP-binding, linker, and pore regions to investigate how the ATP activation signal is transmitted within the trimer and how signals generated by ATP and hyperpolarization converge. Analysis of voltage- and [ATP]-dependent gating in these TTCs showed that: (a) Voltage- and [ATP]-dependent gating of P2X2 requires binding of at least two ATP molecules. (b) D315A mutation in the β-14 strand of the linker region connecting the ATP-binding domains to the pore-forming helices induces two different gating modes; this requires the presence of the D315A mutation in at least two subunits. (c) The T339S mutation in the pore domains of all three subunits abolishes the voltage dependence of P2X2 gating in saturating [ATP], making P2X2 equally active at all membrane potentials. Increasing the number of T339S mutations in the TTC results in gradual changes in the voltage dependence of gating from that of the wild-type channel, suggesting equal and independent contributions of the subunits at the pore level. (d) Voltage- and [ATP]-dependent gating in TTCs differs depending on the location of one D315A relative to one K308A that blocks the ATP binding and downstream signal transmission. (e) Voltage- and [ATP]-dependent gating does not depend on where one T339S is located relative to K308A (or D315A). Our results suggest that each intersubunit ATP-binding signal is directly transmitted on the same subunit to the level of D315 via the domain that contributes K308 to the β-14 strand. The signal subsequently spreads equally to all three subunits at the level of the pore, resulting in symmetric and independent contributions of the three

  17. Signal transmission within the P2X2 trimeric receptor

    PubMed Central

    Kubo, Yoshihiro

    2014-01-01

    P2X2 receptor channel, a homotrimer activated by the binding of extracellular adenosine triphosphate (ATP) to three intersubunit ATP-binding sites (each located ∼50 Å from the ion permeation pore), also shows voltage-dependent activation upon hyperpolarization. Here, we used tandem trimeric constructs (TTCs) harboring critical mutations at the ATP-binding, linker, and pore regions to investigate how the ATP activation signal is transmitted within the trimer and how signals generated by ATP and hyperpolarization converge. Analysis of voltage- and [ATP]-dependent gating in these TTCs showed that: (a) Voltage- and [ATP]-dependent gating of P2X2 requires binding of at least two ATP molecules. (b) D315A mutation in the β-14 strand of the linker region connecting the ATP-binding domains to the pore-forming helices induces two different gating modes; this requires the presence of the D315A mutation in at least two subunits. (c) The T339S mutation in the pore domains of all three subunits abolishes the voltage dependence of P2X2 gating in saturating [ATP], making P2X2 equally active at all membrane potentials. Increasing the number of T339S mutations in the TTC results in gradual changes in the voltage dependence of gating from that of the wild-type channel, suggesting equal and independent contributions of the subunits at the pore level. (d) Voltage- and [ATP]-dependent gating in TTCs differs depending on the location of one D315A relative to one K308A that blocks the ATP binding and downstream signal transmission. (e) Voltage- and [ATP]-dependent gating does not depend on where one T339S is located relative to K308A (or D315A). Our results suggest that each intersubunit ATP-binding signal is directly transmitted on the same subunit to the level of D315 via the domain that contributes K308 to the β-14 strand. The signal subsequently spreads equally to all three subunits at the level of the pore, resulting in symmetric and independent contributions of the three

  18. Emerging roles of P2X receptors in cancer.

    PubMed

    Adinolfi, Elena; Capece, Marina; Amoroso, Francesca; De Marchi, Elena; Franceschini, Alessia

    2015-01-01

    Tumor microenvironment composition strongly conditions cancer growth and progression, acting not only at cancer itself but also modifying its interactions with immune, endothelial and nervous cells. Extracellular ATP and its receptors recently gained increasing attention in the oncological field. ATP accumulates in cancer milieu through spontaneous release, tumor necrosis or chemotherapy exerting a trophic activity on cancer cells, modulating the cross talk among tumor, and surrounding tissues. Accordingly, ATP gated P2X receptors emerged as central players in tumor development, invasion, progression and related symptoms. Indeed, P2X receptors are expressed and are functional not only on tumor cells but also in immune-infiltrate and nearby neurons. In this review, we summarize recent findings on P2X receptors role in tumor cell differentiation, bioenergetics, angiogenesis, metastasis and associated pain, giving an outline of the potential anti-neoplastic activity of receptor agonists and antagonists. PMID:25312206

  19. Reduced vas deferens contraction and male infertility in mice lacking P2X1 receptors.

    PubMed

    Mulryan, K; Gitterman, D P; Lewis, C J; Vial, C; Leckie, B J; Cobb, A L; Brown, J E; Conley, E C; Buell, G; Pritchard, C A; Evans, R J

    2000-01-01

    P2X1 receptors for ATP are ligand-gated cation channels, present on many excitable cells including vas deferens smooth muscle cells. A substantial component of the contractile response of the vas deferens to sympathetic nerve stimulation, which propels sperm into the ejaculate, is mediated through P2X receptors. Here we show that male fertility is reduced by approximately 90% in mice with a targeted deletion of the P2X1 receptor gene. Male mice copulate normally--reduced fertility results from a reduction of sperm in the ejaculate and not from sperm dysfunction. Female mice and heterozygote mice are unaffected. In P2X1-receptor-deficient mice, contraction of the vas deferens to sympathetic nerve stimulation is reduced by up to 60% and responses to P2X receptor agonists are abolished. These results show that P2X1 receptors are essential for normal male reproductive function and suggest that the development of selective P2X1 receptor antagonists may provide an effective non-hormonal male contraceptive pill. Also, agents that potentiate the actions of ATP at P2X1 receptors may be useful in the treatment of male infertility. PMID:10638758

  20. Nociceptive transmission and modulation via P2X receptors in central pain syndrome.

    PubMed

    Kuan, Yung-Hui; Shyu, Bai-Chuang

    2016-01-01

    Painful sensations are some of the most frequent complaints of patients who are admitted to local medical clinics. Persistent pain varies according to its causes, often resulting from local tissue damage or inflammation. Central somatosensory pathway lesions that are not adequately relieved can consequently cause central pain syndrome or central neuropathic pain. Research on the molecular mechanisms that underlie this pathogenesis is important for treating such pain. To date, evidence suggests the involvement of ion channels, including adenosine triphosphate (ATP)-gated cation channel P2X receptors, in central nervous system pain transmission and persistent modulation upon and following the occurrence of neuropathic pain. Several P2X receptor subtypes, including P2X2, P2X3, P2X4, and P2X7, have been shown to play diverse roles in the pathogenesis of central pain including the mediation of fast transmission in the peripheral nervous system and modulation of neuronal activity in the central nervous system. This review article highlights the role of the P2X family of ATP receptors in the pathogenesis of central neuropathic pain and pain transmission. We discuss basic research that may be translated to clinical application, suggesting that P2X receptors may be treatment targets for central pain syndrome. PMID:27230068

  1. Presynaptic P2X1-3 and α3-containing nicotinic receptors assemble into functionally interacting ion channels in the rat hippocampus.

    PubMed

    Rodrigues, Ricardo J; Almeida, Teresa; Díaz-Hernández, Miguel; Marques, Joana M; Franco, Rafael; Solsona, Carles; Miras-Portugal, María Teresa; Ciruela, Francisco; Cunha, Rodrigo A

    2016-06-01

    Previous studies documented a cross-talk between purinergic P2X (P2XR) and nicotinic acetylcholine receptors (nAChR) in heterologous expression systems and peripheral preparations. We now investigated if this occurred in native brain preparations and probed its physiological function. We found that P2XR and nAChR were enriched in hippocampal terminals, where both P2X1-3R and α3, but not α4, nAChR subunits were located in the active zone and in dopamine-β-hydroxylase-positive hippocampal terminals. Notably, P2XR ligands displaced nAChR binding and nAChR ligands displaced P2XR binding to hippocampal synaptosomes. In addition, a negative P2XR/nAChR cross-talk was observed in the control of the evoked release of noradrenaline from rat hippocampal synaptosomes, characterized by a less-than-additive facilitatory effect upon co-activation of both receptors. This activity-dependent cross-inhibition was confirmed in Xenopus oocytes transfected with P2X1-3Rs and α3β2 (but not α4β2) nAChR. Besides, P2X2 co-immunoprecipitated α3β2 (but not α4β2) nAChR, both in HEK cells and rat hippocampal membranes indicating that this functional interaction is supported by a physical association between P2XR and nAChR. Moreover, eliminating extracellular ATP with apyrase in hippocampal slices promoted the inhibitory effect of the nAChR antagonist tubocurarine on noradrenaline release induced by high- but not low-frequency stimulation. Overall, these results provide integrated biochemical, pharmacological and functional evidence showing that P2X1-3R and α3β2 nAChR are physically and functionally interconnected at the presynaptic level to control excessive noradrenergic terminal activation upon intense synaptic firing in the hippocampus. PMID:26801076

  2. P2X7 on Mouse T Cells: One Channel, Many Functions

    PubMed Central

    Rissiek, Björn; Haag, Friedrich; Boyer, Olivier; Koch-Nolte, Friedrich; Adriouch, Sahil

    2015-01-01

    The P2X7 receptor is an adenosine triphosphate (ATP)-gated cation channel that is expressed by several cells of the immune system. P2X7 is best known for its proinflammatory role in promoting inflammasome formation and release of mature interleukin (IL)-1β by innate immune cells. Mounting evidence indicates that P2X7 is also an important regulatory receptor of murine and human T cell functions. Murine T cells express a sensitive splice variant of P2X7 that can be activated either by non-covalent binding of ATP or, in the presence of nicotinamide adenine dinucleotide, by its covalent ADP-ribosylation catalyzed by the ecto-ADP-ribosyltransferase ARTC2.2. Prolonged activation of P2X7 by either one of these pathways triggers the induction of T cell death. Conversely, lower concentrations of ATP can activate P2X7 to enhance T cell proliferation and production of IL-2. In this review, we will highlight the molecular and cellular consequences of P2X7 activation on mouse T cells and its versatile role in T cell homeostasis and activation. Further, we will discuss important differences in the function of P2X7 on human and murine T cells. PMID:26042119

  3. Neuromodulation by extracellular ATP and P2X receptors in the CNS

    PubMed Central

    Khakh, Baljit S.; North, R. Alan

    2014-01-01

    Extracellular adenosine 5’ triphosphate (ATP) is a widespread cell-to-cell signaling molecule in the brain, where it activates cell surface P2X and P2Y receptors. P2X receptors define a protein family unlike other neurotransmitter-gated ion channels in terms of sequence, subunit topology, assembly and architecture. Within milliseconds of binding ATP, they catalyze the opening of a cation-selective pore. However, recent data show that P2X receptors often underlie neuromodulatory responses on slower time scales of seconds or longer. Herein, we review these findings at molecular, cellular and systems levels. We propose that, while P2X receptors are fast ligand-gated cation channels, they are most adept at mediating slow neuromodulatory functions that are more widespread and more physiologically utilized than fast ATP synaptic transmission in the CNS. PMID:23040806

  4. New P2X3 receptor antagonists. Part 1: Discovery and optimization of tricyclic compounds.

    PubMed

    Szántó, Gábor; Makó, Attila; Bata, Imre; Farkas, Bence; Kolok, Sándor; Vastag, Mónika; Cselenyák, Attila

    2016-08-15

    Purinergic P2X3 receptors are trimeric ligand-gated ion channels whose antagonism is an appealing yet challenging and not fully validated drug development idea. With the aim of identification of an orally active, potent human P2X3 receptor antagonist compound that can penetrate the central nervous system, the compound collection of Gedeon Richter was screened. A hit series of tricyclic compounds was subjected to a rapid, two-step optimization process focusing on increasing potency, improving metabolic stability and CNS penetrability. Attempts resulted in compound 65, a potential tool compound for testing P2X3 inhibitory effects in vivo. PMID:27423478

  5. Molecular Structure and Regulation of P2X Receptors With a Special Emphasis on the Role of P2X2 in the Auditory System.

    PubMed

    Mittal, Rahul; Chan, Brandon; Grati, M'hamed; Mittal, Jeenu; Patel, Kunal; Debs, Luca H; Patel, Amit P; Yan, Denise; Chapagain, Prem; Liu, Xue Zhong

    2016-08-01

    The P2X purinergic receptors are cation-selective channels gated by extracellular adenosine 5'-triphosphate (ATP). These purinergic receptors are found in virtually all mammalian cell types and facilitate a number of important physiological processes. Within the past few years, the characterization of crystal structures of the zebrafish P2X4 receptor in its closed and open states has provided critical insights into the mechanisms of ligand binding and channel activation. Understanding of this gating mechanism has facilitated to design and interpret new modeling and structure-function experiments to better elucidate how different agonists and antagonists can affect the receptor with differing levels of potency. This review summarizes the current knowledge on the structure, activation, allosteric modulators, function, and location of the different P2X receptors. Moreover, an emphasis on the P2X2 receptors has been placed in respect to its role in the auditory system. In particular, the discovery of three missense mutations in P2X2 receptors could become important areas of study in the field of gene therapy to treat progressive and noise-induced hearing loss. J. Cell. Physiol. 231: 1656-1670, 2016. © 2015 Wiley Periodicals, Inc. PMID:26627116

  6. Podocyte Purinergic P2X4 Channels Are Mechanotransducers That Mediate Cytoskeletal Disorganization.

    PubMed

    Forst, Anna-Lena; Olteanu, Vlad Sorin; Mollet, Géraldine; Wlodkowski, Tanja; Schaefer, Franz; Dietrich, Alexander; Reiser, Jochen; Gudermann, Thomas; Mederos y Schnitzler, Michael; Storch, Ursula

    2016-03-01

    Podocytes are specialized, highly differentiated epithelial cells in the kidney glomerulus that are exposed to glomerular capillary pressure and possible increases in mechanical load. The proteins sensing mechanical forces in podocytes are unconfirmed, but the classic transient receptor potential channel 6 (TRPC6) interacting with the MEC-2 homolog podocin may form a mechanosensitive ion channel complex in podocytes. Here, we observed that podocytes respond to mechanical stimulation with increased intracellular calcium concentrations and increased inward cation currents. However, TRPC6-deficient podocytes responded in a manner similar to that of control podocytes, and mechanically induced currents were unaffected by genetic inactivation of TRPC1/3/6 or administration of the broad-range TRPC blocker SKF-96365. Instead, mechanically induced currents were significantly decreased by the specific P2X purinoceptor 4 (P2X4) blocker 5-BDBD. Moreover, mechanical P2X4 channel activation depended on cholesterol and podocin and was inhibited by stabilization of the actin cytoskeleton. Because P2X4 channels are not intrinsically mechanosensitive, we investigated whether podocytes release ATP upon mechanical stimulation using a fluorometric approach. Indeed, mechanically induced ATP release from podocytes was observed. Furthermore, 5-BDBD attenuated mechanically induced reorganization of the actin cytoskeleton. Altogether, our findings reveal a TRPC channel-independent role of P2X4 channels as mechanotransducers in podocytes. PMID:26160898

  7. Permeation Properties of a P2X Receptor in the Green Algae Ostreococcus tauri*

    PubMed Central

    Fountain, Samuel J.; Cao, Lishuang; Young, Mark T.; North, R. Alan

    2008-01-01

    We have cloned a P2X receptor (OtP2X) from the green algae Ostreococcus tauri. The 42-kDa receptor shares ∼28% identity with human P2X receptors and 23% with the Dictyostelium P2X receptor. ATP application evoked flickery single channel openings in outside-out membrane patches from human embryonic kidney 293 cells expressing OtP2X. Whole-cell recordings showed concentration-dependent cation currents reversing close to zero mV; ATP gave a half-maximal current at 250 μm. αβ-Methylene-ATP evoked only small currents in comparison to ATP (EC50 > 5 mm). 2′,3′-O-(4-Benzoylbenzoyl)-ATP, βγ-imido-ATP, ADP, and several other nucleotide triphosphates did not activate any current. The currents evoked by 300 μm ATP were not inhibited by 100 μm suramin, pyridoxal-phosphate-6-azophenyl-2′,4′-disulfonic acid, 2′,3′-O-(2,4,6-trinitrophenol)-ATP, or copper. Ion substitution experiments indicated permeabilities relative to sodium with the rank order calcium >choline >Tris >tetraethylammonium >N-methyl-d-glucosamine. However, OtP2X had a low relative calcium permeability (PCa/PNa = 0.4) in comparison with other P2X receptors. This was due at least in part to the presence of an asparagine residue (Asn353) at a position in the second transmembrane domain in place of the aspartate that is completely conserved in all other P2X receptor subunits, because replacement of Asn353 with aspartate increased calcium permeability by ∼50%. The results indicate that the ability of ATP to gate cation permeation across membranes exists in cells that diverged in evolutionary terms from animals about 1 billion years ago. PMID:18381285

  8. Principles and properties of ion flow in P2X receptors

    PubMed Central

    Samways, Damien S. K.; Li, Zhiyuan; Egan, Terrance M.

    2014-01-01

    P2X receptors are a family of trimeric ion channels that are gated by extracellular adenosine 5′-triphosphate (ATP). These receptors have long been a subject of intense research interest by virtue of their vital role in mediating the rapid and direct effects of extracellular ATP on membrane potential and cytosolic Ca2+ concentration, which in turn underpin the ability of ATP to regulate a diverse range of clinically significant physiological functions, including those associated with the cardiovascular, sensory, and immune systems. An important aspect of an ion channel's function is, of course, the means by which it transports ions across the biological membrane. A concerted effort by investigators over the last two decades has culminated in significant advances in our understanding of how P2X receptors conduct the inward flux of Na+ and Ca2+ in response to binding by ATP. However, this work has relied heavily on results from current recordings of P2X receptors altered by site-directed mutagenesis. In the absence of a 3-dimensional channel structure, this prior work provided only a vague and indirect appreciation of the relationship between structure, ion selectivity and flux. The recent publication of the crystal structures for both the closed and open channel conformations of the zebrafish P2X4 receptor has thus proved a significant boon, and has provided an important opportunity to overview the amassed functional data in the context of a working 3-dimensional model of a P2X receptor. In this paper, we will attempt to reconcile the existing functional data regarding ion permeation through P2X receptors with the available crystal structure data, highlighting areas of concordance and discordance as appropriate. PMID:24550775

  9. Principles and properties of ion flow in P2X receptors.

    PubMed

    Samways, Damien S K; Li, Zhiyuan; Egan, Terrance M

    2014-01-01

    P2X receptors are a family of trimeric ion channels that are gated by extracellular adenosine 5'-triphosphate (ATP). These receptors have long been a subject of intense research interest by virtue of their vital role in mediating the rapid and direct effects of extracellular ATP on membrane potential and cytosolic Ca(2+) concentration, which in turn underpin the ability of ATP to regulate a diverse range of clinically significant physiological functions, including those associated with the cardiovascular, sensory, and immune systems. An important aspect of an ion channel's function is, of course, the means by which it transports ions across the biological membrane. A concerted effort by investigators over the last two decades has culminated in significant advances in our understanding of how P2X receptors conduct the inward flux of Na(+) and Ca(2+) in response to binding by ATP. However, this work has relied heavily on results from current recordings of P2X receptors altered by site-directed mutagenesis. In the absence of a 3-dimensional channel structure, this prior work provided only a vague and indirect appreciation of the relationship between structure, ion selectivity and flux. The recent publication of the crystal structures for both the closed and open channel conformations of the zebrafish P2X4 receptor has thus proved a significant boon, and has provided an important opportunity to overview the amassed functional data in the context of a working 3-dimensional model of a P2X receptor. In this paper, we will attempt to reconcile the existing functional data regarding ion permeation through P2X receptors with the available crystal structure data, highlighting areas of concordance and discordance as appropriate. PMID:24550775

  10. Quantifying Ca2+ Current and Permeability in ATP-gated P2X7 Receptors*

    PubMed Central

    Liang, Xin; Samways, Damien S. K.; Wolf, Kyle; Bowles, Elizabeth A.; Richards, Jennifer P.; Bruno, Jonathan; Dutertre, Sébastien; DiPaolo, Richard J.; Egan, Terrance M.

    2015-01-01

    ATP-gated P2X7 receptors are prominently expressed in inflammatory cells and play a key role in the immune response. A major consequence of receptor activation is the regulated influx of Ca2+ through the self-contained cation non-selective channel. Although the physiological importance of the resulting rise in intracellular Ca2+ is universally acknowledged, the biophysics of the Ca2+ flux responsible for the effects are poorly understood, largely because traditional methods of measuring Ca2+ permeability are difficult to apply to P2X7 receptors. Here we use an alternative approach, called dye-overload patch-clamp photometry, to quantify the agonist-gated Ca2+ flux of recombinant P2X7 receptors of dog, guinea pig, human, monkey, mouse, rat, and zebrafish. We find that the magnitude of the Ca2+ component of the ATP-gated current depends on the species of origin, the splice variant, and the concentration of the purinergic agonist. We also measured a significant contribution of Ca2+ to the agonist-gated current of the native P2X7Rs of mouse and human immune cells. Our results provide cross-species quantitative measures of the Ca2+ current of the P2X7 receptor for the first time, and suggest that the cytoplasmic N terminus plays a meaningful role in regulating the flow of Ca2+ through the channel. PMID:25645917

  11. New insights of P2X7 receptor signaling pathway in alveolar functions.

    PubMed

    Mishra, Amarjit

    2013-01-01

    Purinergic P2X7 receptor (P2X7R), an ATP-gated cation channel, is unique among all other family members because of its ability to respond to various stimuli and to modulate pro-inflammatory signaling. The activation of P2X7R in immune cells is absolutely required for mature interleukin -1beta (IL-1beta) and IL-18 production and release. Lung alveoli are lined by the structural alveolar epithelial type I (AEC I) and alveolar epithelial type II cells (AEC II). AEC I plays important roles in alveolar barrier protection and fluid homeostasis whereas AEC II synthesizes and secrete surfactant and prevents alveoli from collapse. Earlier studies indicated that purinergic P2X7 receptors were specifically expressed in AEC I. However, their implication in alveolar functions has not been explored. This paper reviews two important signaling pathways of P2X7 receptors in surfactant homeostatsis and Acute Lung Injury (ALI). Thus, P2X7R resides at the critical nexus of alveolar pathophysiology. PMID:23634990

  12. Single Channel Properties of P2X2 Purinoceptors

    PubMed Central

    Ding, Shinghua; Sachs, Frederick

    1999-01-01

    The single channel properties of cloned P2X2 purinoceptors expressed in human embryonic kidney (HEK) 293 cells and Xenopus oocytes were studied in outside-out patches. The mean single channel current–voltage relationship exhibited inward rectification in symmetric solutions with a chord conductance of ∼30 pS at −100 mV in 145 mM NaCl. The channel open state exhibited fast flickering with significant power beyond 10 kHz. Conformational changes, not ionic blockade, appeared responsible for the flickering. The equilibrium constant of Na+ binding in the pore was ∼150 mM at 0 mV and voltage dependent. The binding site appeared to be ∼0.2 of the electrical distance from the extracellular surface. The mean channel current and the excess noise had the selectivity: K+ > Rb+ > Cs+ > Na+ > Li+. ATP increased the probability of being open (Po) to a maximum of 0.6 with an EC50 of 11.2 μM and a Hill coefficient of 2.3. Lowering extracellular pH enhanced the apparent affinity of the channel for ATP with a pKa of ∼7.9, but did not cause a proton block of the open channel. High pH slowed the rise time to steps of ATP without affecting the fall time. The mean single channel amplitude was independent of pH, but the excess noise increased with decreasing pH. Kinetic analysis showed that ATP shortened the mean closed time but did not affect the mean open time. Maximum likelihood kinetic fitting of idealized single channel currents at different ATP concentrations produced a model with four sequential closed states (three binding steps) branching to two open states that converged on a final closed state. The ATP association rates increased with the sequential binding of ATP showing that the binding sites are not independent, but positively cooperative. Partially liganded channels do not appear to open. The predicted Po vs. ATP concentration closely matches the single channel current dose–response curve. PMID:10228183

  13. Characterization of protoberberine analogs employed as novel human P2X{sub 7} receptor antagonists

    SciTech Connect

    Lee, Ga Eun; Lee, Won-Gil; Lee, Song-Yi; Lee, Cho-Rong; Park, Chul-Seung; Chang, Sunghoe; Park, Sung-Gyoo; Song, Mi-Ryoung; Kim, Yong-Chul

    2011-04-15

    The P2X{sub 7} receptor (P2X{sub 7}R), a member of the ATP-gated ion channel family, is regarded as a promising target for therapy of immune-related diseases including rheumatoid arthritis and chronic pain. A group of novel protoberberine analogs (compounds 3-5), discovered by screening of chemical libraries, was here investigated with respect to their function as P2X{sub 7}R antagonists. Compounds 3-5 non-competitively inhibited BzATP-induced ethidium ion influx into hP2X{sub 7}-expressing HEK293 cells, with IC{sub 50} values of 100-300 nM. This antagonistic action on the channel further confirmed that both BzATP-induced inward currents and Ca{sup 2+} influx were strongly inhibited by compounds 3-5 in patch-clamp and Ca{sup 2+} influx assays. The antagonists also effectively suppressed downstream signaling of P2X{sub 7} receptors including IL-1{beta} release and phosphorylation of ERK1/2 and p38 proteins in hP2X{sub 7}-expressing HEK293 cells or in differentiated human monocytes (THP-1 cells). Moreover, IL-2 secretion from CD3/CD28-stimulated Jurkat T cell was also dramatically inhibited by the antagonist. These results imply that novel protoberberine analogs may modulate P2X{sub 7} receptor-mediated immune responses by allosteric inhibition of the receptor. - Graphical abstract: Display Omitted

  14. Co-Expression of Wild-Type P2X7R with Gln460Arg Variant Alters Receptor Function

    PubMed Central

    Aprile-Garcia, Fernando; Metzger, Michael W.; Paez-Pereda, Marcelo; Stadler, Herbert; Acuña, Matías; Liberman, Ana C.; Senin, Sergio A.; Gerez, Juan; Hoijman, Esteban; Refojo, Damian; Mitkovski, Mišo; Panhuysen, Markus; Stühmer, Walter; Holsboer, Florian; Deussing, Jan M.; Arzt, Eduardo

    2016-01-01

    The P2X7 receptor is a member of the P2X family of ligand-gated ion channels. A single-nucleotide polymorphism leading to a glutamine (Gln) by arginine (Arg) substitution at codon 460 of the purinergic P2X7 receptor (P2X7R) has been associated with mood disorders. No change in function (loss or gain) has been described for this SNP so far. Here we show that although the P2X7R-Gln460Arg variant per se is not compromised in its function, co-expression of wild-type P2X7R with P2X7R-Gln460Arg impairs receptor function with respect to calcium influx, channel currents and intracellular signaling in vitro. Moreover, co-immunoprecipitation and FRET studies show that the P2X7R-Gln460Arg variant physically interacts with P2X7R-WT. Specific silencing of either the normal or polymorphic variant rescues the heterozygous loss of function phenotype and restores normal function. The described loss of function due to co-expression, unique for mutations in the P2RX7 gene so far, explains the mechanism by which the P2X7R-Gln460Arg variant affects the normal function of the channel and may represent a mechanism of action for other mutations. PMID:26986975

  15. The Role of the P2X7 Receptor in Infectious Diseases

    PubMed Central

    Miller, Catherine M.; Boulter, Nicola R.; Fuller, Stephen J.; Zakrzewski, Alana M.; Lees, Michael P.; Saunders, Bernadette M.; Wiley, James S.; Smith, Nicholas C.

    2011-01-01

    ATP is an extracellular signal for the immune system, particularly during an inflammatory response. It is sensed by the P2X7 receptor, the expression of which is upregulated by pro-inflammatory cytokines. Activation of the P2X7 receptor opens a cation-specific channel that alters the ionic environment of the cell, activating several pathways, including (i) the inflammasome, leading to production of IL-1β and IL-18; (ii) the stress-activated protein kinase pathway, resulting in apoptosis; (iii) the mitogen-activated protein kinase pathway, leading to generation of reactive oxygen and nitrogen intermediates; and (iv) phospholipase D, stimulating phagosome-lysosome fusion. The P2X7 receptor can initiate host mechanisms to remove pathogens, most particularly those that parasitise macrophages. At the same time, the P2X7 receptor may be subverted by pathogens to modulate host responses. Moreover, recent genetic studies have demonstrated significant associations between susceptibility or resistance to parasites and bacteria, and loss-of-function or gain-of-function polymorphisms in the P2X7 receptor, underscoring its importance in infectious disease. PMID:22102807

  16. Characterisation of ATP analogues to cross-link and label P2X receptors

    PubMed Central

    Agboh, Kelvin C.; Powell, Andrew J.; Evans, Richard J.

    2009-01-01

    P2X receptors are a distinct family of ATP-gated ion channels with a number of physiological roles ranging from smooth muscle contractility to the regulation of blood clotting. In this study we determined whether the UV light-reactive ATP analogues 2-azido ATP, ATP azidoanilide (ATP-AA) and 2′,3′-O-(4-benzoylbenzoyl)-ATP (BzATP) can be used to label the ATP binding site of P2X1 receptors. These analogues were agonists, and in patch clamp studies evoked inward currents from HEK293 cells stably expressing the P2X1 receptor. Following irradiation in the presence of these compounds subsequent responses to an EC50 concentration of ATP were reduced by >65%. These effects were partially reversed by co-application of ATP or suramin with the photo-reactive ATP analogue at the time of irradiation. In autoradiographic studies radiolabelled 2-azido [γ32P] ATP and ATP-AA-[γ32P] cross-linked to P2X1 receptors and this binding was reduced by co-incubation with ATP. These studies demonstrate that photo-reactive ATP analogues can be used to label P2X receptor and may prove useful in elucidating the ATP binding site at this novel class of ATP binding proteins. PMID:18599093

  17. [Peptide components of Geolycosa spider venom modulate P2X receptor activity of rat sensory neurons].

    PubMed

    Savchenko, H A; Vasylevs'kyĭ, A A; Pluzhnykov, K A; Korol'kova, Iu V; Mamenko, M V; Volkova, T M; Maksymiuk, O P; Boĭchuk, Ia A; Hrishyn, Ie V; Kryshtal', O O

    2009-01-01

    Almost each natural venom comprises a considerable combinatorial library of bioactive substances that have been optimized during evolution. Particular attention is devoted currently on a search for new modulators of ion channels from the venoms of arthropods. We have studied the effect of peptidous compounds of the Lycosa spider venom on the activity of P2X receptors in DRG neurons of rats. As a result, at least 7 proteins modulating various P2X receptor-operated ionic currents in the sensory neurons of rats have been found. PMID:19526843

  18. Activation of P2X7 receptors in glial satellite cells reduces pain through downregulation of P2X3 receptors in nociceptive neurons

    PubMed Central

    Chen, Yong; Zhang, Xiaofei; Wang, Congying; Li, GuangWen; Gu, Yanping; Huang, Li-Yen Mae

    2008-01-01

    Purinergic ionotropic P2X7 receptors (P2X7Rs) are closely associated with excitotoxicity and nociception. Inhibition of P2X7R activation has been considered as a potentially useful strategy to improve recovery from spinal cord injury and reduce inflammatory damage to trauma. The physiological functions of P2X7Rs, however, are poorly understood, even though such information is essential for making the P2X7R an effective therapeutic target. We show here that P2X7Rs in satellite cells of dorsal root ganglia tonically inhibit the expression of P2X3Rs in neurons. Reducing P2X7R expression using siRNA or blocking P2X7R activity by antagonists elicits P2X3R up-regulation, increases the activity of sensory neurons responding to painful stimuli, and evokes abnormal nociceptive behaviors in rats. Thus, contrary to the notion that P2X7R activation is cytotoxic, P2X7Rs in satellite cells play a crucial role in maintaining proper P2X3R expression in dorsal root ganglia. Studying the mechanism underlying the P2X7R–P2X3R control, we demonstrate that activation of P2X7Rs evokes ATP release from satellite cells. ATP in turn stimulates P2Y1 receptors in neurons. P2Y1 receptor activation appears to be necessary and sufficient for the inhibitory control of P2X3R expression. We further determine the roles of the P2X7R–P2Y1–P2X3R inhibitory control under injurious conditions. Activation of the inhibitory control effectively prevents the development of allodynia and increases the potency of systemically administered P2X7R agonists in inflamed rats. Thus, direct blocking P2X7Rs, as proposed before, may not be the best strategy for reducing pain or lessening neuronal degeneration because it also disrupts the protective function of P2X7Rs. PMID:18946042

  19. Anthraquinone emodin inhibits human cancer cell invasiveness by antagonizing P2X7 receptors.

    PubMed

    Jelassi, Bilel; Anchelin, Monique; Chamouton, Julie; Cayuela, María Luisa; Clarysse, Lucie; Li, Junying; Goré, Jacques; Jiang, Lin-Hua; Roger, Sébastien

    2013-07-01

    The adenosine 5'-triphosphate (ATP)-gated Ca(2+)-permeable channel P2X7 receptor (P2X7R) is strongly upregulated in many tumors and cancer cells, and has an important role in cancer cell invasion associated with metastases. Emodin (1,3,8-trihydroxy-6-methylanthraquinone) is an anthraquinone derivative originally isolated from Rheum officinale Baill known for decades to possess anticancer properties. In this study, we examined the effects of emodin on P2X7R-dependent Ca(2+) signaling, extracellular matrix degradation, and in vitro and in vivo cancer cell invasiveness using highly aggressive human cancer cells. Inclusion of emodin at doses ≤10 µM in cell culture had no or very mild effect on the cell viability. ATP elicited increases in intracellular Ca(2+) concentration were reduced by 35 and 60% by 1 and 10 µM emodin, respectively. Emodin specifically inhibited P2X7R-mediated currents with an IC50 of 3 µM and did not inhibit the currents mediated by the other human P2X receptors heterologously expressed in human embryonic kidney (HEK293T) cells. ATP-induced increase in gelatinolytic activity, in cancer cell invasiveness in vitro and in cell morphology changes were prevented by 1 µM emodin. Furthermore, such ATP-evoked effects and inhibition by emodin were almost completely ablated in cancer cells transfected with P2X7R-specific small interfering RNA (siRNA) but not with scrambled siRNA. Finally, the in vivo invasiveness of the P2X7R-positive MDA-MB-435s breast cancer cells, assessed using a zebrafish model of micrometastases, was suppressed by 40 and 50% by 1 and 10 µM emodin. Taken together, these results provide consistent evidence to indicate that emodin inhibits human cancer cell invasiveness by specifically antagonizing the P2X7R. PMID:23524196

  20. Effects of antidepressants on P2X7 receptors.

    PubMed

    Wang, Wei; Xiang, Zheng-Hua; Jiang, Chun-Lei; Liu, Wei-Zhi; Shang, Zhi-Lei

    2016-08-30

    Antidepressants including paroxetine, fluoxetine and desipramine are commonly used for treating depression. P2×7 receptors are member of the P2X family. Recent studies indicate that these receptors may constitute a novel potential target for the treatment of depression. In the present study, we examined the action of these antidepressants on cloned rat P2×7 receptors that were stably expressed in human embryonic kidney (HEK) 293 cells by using the whole-cell patch-clamp technique, and found that paroxetine at a dose of 10µM could significantly reduce the inward currents evoked by the P2×7 receptors agonist BzATP by pre-incubation for 6-12 but not by acute application (10µM) or pre-incubation for 2-6h at a dose of 1µM, 3µM or 10µM paroxetine. Neither fluoxetine nor desipramine had significant effects on currents evoked by BzATP either applied acutely or by pre-incubation at various concentrations. These results suggest that the sensitivity of rat P2×7 receptors to antidepressants is different, which may represent an unknown mechanism by which these drugs exert their therapeutic effects and side effects. PMID:27318632

  1. Pharmacological characterization of a novel centrally permeable P2X7 receptor antagonist: JNJ-47965567

    PubMed Central

    Bhattacharya, Anindya; Wang, Qi; Ao, Hong; Shoblock, James R; Lord, Brian; Aluisio, Leah; Fraser, Ian; Nepomuceno, Diane; Neff, Robert A; Welty, Natalie; Lovenberg, Timothy W; Bonaventure, Pascal; Wickenden, Alan D; Letavic, Michael A

    2013-01-01

    BACKGROUND AND PURPOSE An increasing body of evidence suggests that the purinergic receptor P2X, ligand-gated ion channel, 7 (P2X7) in the CNS may play a key role in neuropsychiatry, neurodegeneration and chronic pain. In this study, we characterized JNJ-47965567, a centrally permeable, high-affinity, selective P2X7 antagonist. EXPERIMENTAL APPROACH We have used a combination of in vitro assays (calcium flux, radioligand binding, electrophysiology, IL-1β release) in both recombinant and native systems. Target engagement of JNJ-47965567 was demonstrated by ex vivo receptor binding autoradiography and in vivo blockade of Bz-ATP induced IL-1β release in the rat brain. Finally, the efficacy of JNJ-47965567 was tested in standard models of depression, mania and neuropathic pain. KEY RESULTS JNJ-47965567 is potent high affinity (pKi 7.9 ± 0.07), selective human P2X7 antagonist, with no significant observed speciation. In native systems, the potency of the compound to attenuate IL-1β release was 6.7 ± 0.07 (human blood), 7.5 ± 0.07 (human monocytes) and 7.1 ± 0.1 (rat microglia). JNJ-47965567 exhibited target engagement in rat brain, with a brain EC50 of 78 ± 19 ng·mL−1 (P2X7 receptor autoradiography) and functional block of Bz-ATP induced IL-1β release. JNJ-47965567 (30 mg·kg−1) attenuated amphetamine-induced hyperactivity and exhibited modest, yet significant efficacy in the rat model of neuropathic pain. No efficacy was observed in forced swim test. Conclusion and Implications JNJ-47965567 is centrally permeable, high affinity P2X7 antagonist that can be used to probe the role of central P2X7 in rodent models of CNS pathophysiology. PMID:23889535

  2. P2X receptors in cochlear Deiters' cells

    PubMed Central

    Chen, Chu; Bobbin, Richard P

    1998-01-01

    The ionotropic purinoceptors in isolated Deiters' cells of guinea-pig cochlea were characterized by use of the whole-cell variant of the patch-clamp technique.Extracellular application of adenosine 5′-triphosphate (ATP) induced a dose-dependent inward current when the cells were voltage-clamped at −80 mV. The ATP-induced current showed desensitization and had a reversal potential around −4 mV.Increasing intracellular free Ca2+ by decreasing the concentration of EGTA in the pipette solution reduced the amplitude of the ATP-gated current.The order of agonist potency was: 2-methylthioATP (2-meSATP)>ATP>benzoylbenzoyl-ATP (BzATP)>α,β-methyleneATP (α,β,meATP>adenosine 5′-diphosphate (ADP)>uridine 5′-triphosphate (UTP)>adenosine 5′-monophosphate (AMP)=adenosine (Ad).Pretreatment with forskolin (10 μM), 8-bromoadenosine-3′,5′-cyclophosphate (8-Br-cyclic AMP, 1 mM), 3-isobutyl-1-methylxanthine (IBMX, 1 mM) or phorbol-12-myristate-13-acetate (PMA, 1 μM) reversibly reduced the ATP-induced peak current.The results are consistent with molecular biological data which indicate that P2X2 purinoceptors are present in Deiters' cells. In addition, the reduction of the ATP-gated current by activators of protein kinase A and protein kinase C indicates that these P2X2 purinoceptors can be functionally modulated by receptor phosphorylation. PMID:9641551

  3. Critical Evaluation of P2X7 Receptor Antagonists in Selected Seizure Models

    PubMed Central

    Fischer, Wolfgang; Franke, Heike; Krügel, Ute; Müller, Heiko; Dinkel, Klaus; Lord, Brian; Letavic, Michael A.; Henshall, David C.; Engel, Tobias

    2016-01-01

    The ATP-gated P2X7 receptor (P2X7R) is a non-selective cation channel which senses high extracellular ATP concentrations and has been suggested as a target for the treatment of neuroinflammation and neurodegenerative diseases. The use of P2X7R antagonists may therefore be a viable approach for treating CNS pathologies, including epileptic disorders. Recent studies showed anticonvulsant potential of P2X7R antagonists in certain animal models. To extend this work, we tested three CNS-permeable P2X7R blocker (Brilliant Blue G, AFC-5128, JNJ-47965567) and a natural compound derivative (tanshinone IIA sulfonate) in four well-characterized animal seizure models. In the maximal electroshock seizure threshold test and the pentylenetetrazol (PTZ) seizure threshold test in mice, none of the four compounds demonstrated anticonvulsant effects when given alone. Notably, in combination with carbamazepine, both AFC-5128 and JNJ-47965567 increased the threshold in the maximal electroshock seizure test. In the PTZ-kindling model in rats, useful for testing antiepileptogenic activities, Brilliant Blue G and tanshinone exhibited a moderate retarding effect, whereas the potent P2X7R blocker AFC-5128 and JNJ-47965567 showed a significant and long-lasting delay in kindling development. In fully kindled rats, the investigated compounds revealed modest effects to reduce the mean seizure stage. Furthermore, AFC-5128- and JNJ-47965567-treated animals displayed strongly reduced Iba 1 and GFAP immunoreactivity in the hippocampal CA3 region. In summary, our results show that P2X7R antagonists possess no remarkable anticonvulsant effects in the used acute screening tests, but can attenuate chemically-induced kindling. Further studies would be of interest to support the concept that P2X7R signalling plays a crucial role in the pathogenesis of epileptic disorders. PMID:27281030

  4. P2X1 Receptor Antagonists Inhibit HIV-1 Fusion by Blocking Virus-Coreceptor Interactions

    PubMed Central

    Giroud, Charline; Marin, Mariana; Hammonds, Jason; Spearman, Paul

    2015-01-01

    ABSTRACT HIV-1 Env glycoprotein-mediated fusion is initiated upon sequential binding of Env to CD4 and the coreceptor CXCR4 or CCR5. Whereas these interactions are thought to be necessary and sufficient to promote HIV-1 fusion, other host factors can modulate this process. Previous studies reported potent inhibition of HIV-1 fusion by selective P2X1 receptor antagonists, including NF279, and suggested that these receptors play a role in HIV-1 entry. Here we investigated the mechanism of antiviral activity of NF279 and found that this compound does not inhibit HIV-1 fusion by preventing the activation of P2X1 channels but effectively blocks the binding of the virus to CXCR4 or CCR5. The notion of an off-target effect of NF279 on HIV-1 fusion is supported by the lack of detectable expression of P2X1 receptors in cells used in fusion experiments and by the fact that the addition of ATP or the enzymatic depletion of ATP in culture medium does not modulate viral fusion. Importantly, NF279 fails to inhibit HIV-1 fusion with cell lines and primary macrophages when added at an intermediate stage downstream of Env-CD4-coreceptor engagement. Conversely, in the presence of NF279, HIV-1 fusion is arrested downstream of CD4 binding but prior to coreceptor engagement. NF279 also antagonizes the signaling function of CCR5, CXCR4, and another chemokine receptor, as evidenced by the suppression of calcium responses elicited by specific ligands and by recombinant gp120. Collectively, our results demonstrate that NF279 is a dual HIV-1 coreceptor inhibitor that interferes with the functional engagement of CCR5 and CXCR4 by Env. IMPORTANCE Inhibition of P2X receptor activity suppresses HIV-1 fusion and replication, suggesting that P2X signaling is involved in HIV-1 entry. However, mechanistic experiments conducted in this study imply that P2X1 receptor is not expressed in target cells or involved in viral fusion. Instead, we found that inhibition of HIV-1 fusion by a specific P2X1

  5. P2X7 receptor activation regulates rapid unconventional export of transglutaminase-2

    PubMed Central

    Adamczyk, Magdalena; Griffiths, Rhiannon; Dewitt, Sharon; Knäuper, Vera; Aeschlimann, Daniel

    2015-01-01

    ABSTRACT Transglutaminases (denoted TG or TGM) are externalized from cells via an unknown unconventional secretory pathway. Here, we show for the first time that purinergic signaling regulates active secretion of TG2 (also known as TGM2), an enzyme with a pivotal role in stabilizing extracellular matrices and modulating cell–matrix interactions in tissue repair. Extracellular ATP promotes TG2 secretion by macrophages, and this can be blocked by a selective antagonist against the purinergic receptor P2X7 (P2X7R, also known as P2RX7). Introduction of functional P2X7R into HEK293 cells is sufficient to confer rapid, regulated TG2 export. By employing pharmacological agents, TG2 release could be separated from P2X7R-mediated microvesicle shedding. Neither Ca2+ signaling alone nor membrane depolarization triggered TG2 secretion, which occurred only upon receptor membrane pore formation and without pannexin channel involvement. A gain-of-function mutation in P2X7R associated with autoimmune disease caused enhanced TG2 externalization from cells, and this correlated with increased pore activity. These results provide a mechanistic explanation for a link between active TG2 secretion and inflammatory responses, and aberrant enhanced TG2 activity in certain autoimmune conditions. PMID:26542019

  6. Key Sites for P2X Receptor Function and Multimerization: Overview of Mutagenesis Studies on a Structural Basis

    PubMed Central

    Hausmann, Ralf; Kless, Achim; Schmalzing, Günther

    2015-01-01

    P2X receptors constitute a seven-member family (P2X1-7) of extracellular ATP-gated cation channels of widespread expression. Because P2X receptors have been implicated in neurological, inflammatory and cardiovascular diseases, they constitute promising drug targets. Since the first P2X cDNA sequences became available in 1994, numerous site-directed mutagenesis studies have been conducted to disclose key sites of P2X receptor function and oligomerization. The publication of the 3-Å crystal structures of the zebrafish P2X4 (zfP2X4) receptor in the homotrimeric apo-closed and ATP-bound open states in 2009 and 2012, respectively, has ushered a new era by allowing for the interpretation of the wealth of molecular data in terms of specific three-dimensional models and by paving the way for designing more-decisive experiments. Thanks to these structures, the last five years have provided invaluable insight into our understanding of the structure and function of the P2X receptor class of ligandgated ion channels. In this review, we provide an overview of mutagenesis studies of the pre- and post-crystal structure eras that identified amino acid residues of key importance for ligand binding, channel gating, ion flow, formation of the pore and the channel gate, and desensitization. In addition, the sites that are involved in the trimerization of P2X receptors are reviewed based on mutagenesis studies and interface contacts that were predicted by the zfP2X4 crystal structures. PMID:25439586

  7. A P2X receptor from the tardigrade species Hypsibius dujardini with fast kinetics and sensitivity to zinc and copper

    PubMed Central

    Bavan, Selvan; Straub, Volko A; Blaxter, Mark L; Ennion, Steven J

    2009-01-01

    Background Orthologs of the vertebrate ATP gated P2X channels have been identified in Dictyostelium and green algae, demonstrating that the emergence of ionotropic purinergic signalling was an early event in eukaryotic evolution. However, the genomes of a number of animals including Drosophila melanogaster and Caenorhabditis elegans, both members of the Ecdysozoa superphylum, lack P2X-like proteins, whilst other species such as the flatworm Schistosoma mansoni have P2X proteins making it unclear as to what stages in evolution P2X receptors were lost. Here we describe the functional characterisation of a P2X receptor (HdP2X) from the tardigrade Hypsibius dujardini demonstrating that purinergic signalling is preserved in some ecdysozoa. Results ATP (EC50 ~44.5 μM) evoked transient inward currents in HdP2X with millisecond rates of activation and desensitisation. HdP2X is antagonised by pyridoxal-phosphate-6-azophenyl-2',4' disulfonic acid (IC50 15.0 μM) and suramin (IC50 22.6 μM) and zinc and copper inhibit ATP-evoked currents with IC50 values of 62.8 μM and 19.9 μM respectively. Site-directed mutagenesis showed that unlike vertebrate P2X receptors, extracellular histidines do not play a major role in coordinating metal binding in HdP2X. However, H306 was identified as playing a minor role in the actions of copper but not zinc. Ivermectin potentiated responses to ATP with no effect on the rates of current activation or decay. Conclusion The presence of a P2X receptor in a tardigrade species suggests that both nematodes and arthropods lost their P2X genes independently, as both traditional and molecular phylogenies place the divergence between Nematoda and Arthropoda before their divergence from Tardigrada. The phylogenetic analysis performed in our study also clearly demonstrates that the emergence of the family of seven P2X channels in human and other mammalian species was a relatively recent evolutionary event that occurred subsequent to the split between

  8. Impaired P2X1 Receptor-Mediated Adhesion in Eosinophils from Asthmatic Patients.

    PubMed

    Wright, Adam; Mahaut-Smith, Martyn; Symon, Fiona; Sylvius, Nicolas; Ran, Shaun; Bafadhel, Mona; Muessel, Michelle; Bradding, Peter; Wardlaw, Andrew; Vial, Catherine

    2016-06-15

    Eosinophils play an important role in the pathogenesis of asthma and can be activated by extracellular nucleotides released following cell damage or inflammation. For example, increased ATP concentrations were reported in bronchoalveolar lavage fluids of asthmatic patients. Although eosinophils are known to express several subtypes of P2 receptors for extracellular nucleotides, their function and contribution to asthma remain unclear. In this article, we show that transcripts for P2X1, P2X4, and P2X5 receptors were expressed in healthy and asthmatic eosinophils. The P2X receptor agonist α,β-methylene ATP (α,β-meATP; 10 μM) evoked rapidly activating and desensitizing inward currents (peak 18 ± 3 pA/pF at -60 mV) in healthy eosinophils, typical of P2X1 homomeric receptors, which were abolished by the selective P2X1 antagonist NF449 (1 μM) (3 ± 2 pA/pF). α,β-meATP-evoked currents were smaller in eosinophils from asthmatic patients (8 ± 2 versus 27 ± 5 pA/pF for healthy) but were enhanced following treatment with a high concentration of the nucleotidase apyrase (17 ± 5 pA/pF for 10 IU/ml and 11 ± 3 pA/pF for 0.32 IU/ml), indicating that the channels are partially desensitized by extracellular nucleotides. α,β-meATP (10 μM) increased the expression of CD11b activated form in eosinophils from healthy, but not asthmatic, donors (143 ± 21% and 108 ± 11% of control response, respectively). Furthermore, α,β-meATP increased healthy (18 ± 2% compared with control 10 ± 1%) but not asthmatic (13 ± 1% versus 10 ± 0% for control) eosinophil adhesion. Healthy human eosinophils express functional P2X1 receptors whose activation leads to eosinophil αMβ2 integrin-dependent adhesion. P2X1 responses are constitutively reduced in asthmatic compared with healthy eosinophils, probably as the result of an increase in extracellular nucleotide concentration. PMID:27183585

  9. The P2X1 receptor is required for neutrophil extravasation during lipopolysaccharide-induced lethal endotoxemia in mice.

    PubMed

    Maître, Blandine; Magnenat, Stéphanie; Heim, Véronique; Ravanat, Catherine; Evans, Richard J; de la Salle, Henri; Gachet, Christian; Hechler, Béatrice

    2015-01-15

    Extracellular ATP is becoming increasingly recognized as an important regulator of inflammation. However, the known repertoire of P2 receptor subtypes responsible for the proinflammatory effects of ATP is sparse. We looked at whether the P2X1 receptor, an ATP-gated cation channel present on platelets, neutrophils, and macrophages, participates in the acute systemic inflammation provoked by LPS. Compared with wild-type (WT) mice, P2X1(-/-) mice displayed strongly diminished pathological responses, with dampened neutrophil accumulation in the lungs, less tissue damage, reduced activation of coagulation, and resistance to LPS-induced death. P2X1 receptor deficiency also was associated with a marked reduction in plasma levels of the main proinflammatory cytokines and chemokines induced by LPS. Interestingly, macrophages and neutrophils isolated from WT and P2X1(-/-) mice produced similar levels of proinflammatory cytokines when stimulated with LPS in vitro. Intravital microscopy revealed a defect in LPS-induced neutrophil emigration from cremaster venules into the tissues of P2X1(-/-) mice. Using adoptive transfer of immunofluorescently labeled neutrophils from WT and P2X1(-/-) mice into WT mice, we demonstrate that the absence of the P2X1 receptor on neutrophils was responsible for this defect. This study reveals a major role for the P2X1 receptor in LPS-induced lethal endotoxemia through its critical involvement in neutrophil emigration from venules. PMID:25480563

  10. A potential therapeutic role for P2X7 receptor (P2X7R) antagonists in the treatment of inflammatory diseases

    PubMed Central

    Arulkumaran, Nishkantha; Unwin, Robert J.; Tam, Frederick W. K.

    2011-01-01

    Introduction The P2X7 receptor (P2X7R) has an important role in inflammation and immunity, but until recently, clinical application has been limited by a lack of specific antagonists. Recent studies using P2X7R knockout (KO) mice and specific receptor antagonists have shown that the P2X7R is an important therapeutic target in inflammatory diseases. Areas covered We have reviewed the current literature on the role of the P2X7R in inflammatory diseases, focusing on potential therapeutic applications of selective P2X7R antagonists as an anti-inflammatory agent. Particular emphasis has been placed on the potential role of P2X7R in common inflammatory diseases. The latest developments in phase I and II clinical trials of P2X7R antagonists are covered. Expert opinion Recent studies using gene KO mice and selective P2X7R antagonists suggest that P2X7R is a viable therapeutic target for inflammatory diseases. However, efficacious P2X7R antagonists for use in clinical studies are still at an early stage of development. Future challenges include: identifying potential toxicity and side effects of treatment, timing of treatment initiation and its duration in chronic inflammatory conditions, optimum dosage, and development of a functional assay for P2X7R that would help to guide treatment. PMID:21510825

  11. Effect of P2X7 receptor knockout on AQP-5 expression of type I alveolar epithelial cells.

    PubMed

    Ebeling, Georg; Bläsche, Robert; Hofmann, Falk; Augstein, Antje; Kasper, Michael; Barth, Kathrin

    2014-01-01

    P2X7 receptors, ATP-gated cation channels, are specifically expressed in alveolar epithelial cells. The pathophysiological function of this lung cell type, except a recently reported putative involvement in surfactant secretion, is unknown. In addition, P2X7 receptor-deficient mice show reduced inflammation and lung fibrosis after exposure with bleomycin. To elucidate the role of the P2X7 receptor in alveolar epithelial type I cells we characterized the pulmonary phenotype of P2X7 receptor knockout mice by using immunohistochemistry, western blot analysis and real-time RT PCR. No pathomorphological signs of fibrosis were found. Results revealed, however, a remarkable loss of aquaporin-5 protein and mRNA in young knockout animals. Additional in vitro experiments with bleomycin treated precision cut lung slices showed a greater sensitivity of the P2X7 receptor knockout mice in terms of aquaporin-5 reduction as wild type animals. Finally, P2X7 receptor function was examined by using the alveolar epithelial cell lines E10 and MLE-12 for stimulation experiments with bleomycin. The in vitro activation of P2X7 receptor was connected with an increase of aquaporin-5, whereas the inhibition of the receptor with oxidized ATP resulted in down regulation of aquaporin-5. The early loss of aquaporin-5 which can be found in different pulmonary fibrosis models does not implicate a specific pathogenetic role during fibrogenesis. PMID:24941004

  12. Effect of P2X7 Receptor Knockout on AQP-5 Expression of Type I Alveolar Epithelial Cells

    PubMed Central

    Ebeling, Georg; Bläsche, Robert; Hofmann, Falk; Augstein, Antje; Kasper, Michael; Barth, Kathrin

    2014-01-01

    P2X7 receptors, ATP-gated cation channels, are specifically expressed in alveolar epithelial cells. The pathophysiological function of this lung cell type, except a recently reported putative involvement in surfactant secretion, is unknown. In addition, P2X7 receptor-deficient mice show reduced inflammation and lung fibrosis after exposure with bleomycin. To elucidate the role of the P2X7 receptor in alveolar epithelial type I cells we characterized the pulmonary phenotype of P2X7 receptor knockout mice by using immunohistochemistry, western blot analysis and real-time RT PCR. No pathomorphological signs of fibrosis were found. Results revealed, however, a remarkable loss of aquaporin-5 protein and mRNA in young knockout animals. Additional in vitro experiments with bleomycin treated precision cut lung slices showed a greater sensitivity of the P2X7 receptor knockout mice in terms of aquaporin-5 reduction as wild type animals. Finally, P2X7 receptor function was examined by using the alveolar epithelial cell lines E10 and MLE-12 for stimulation experiments with bleomycin. The in vitro activation of P2X7 receptor was connected with an increase of aquaporin-5, whereas the inhibition of the receptor with oxidized ATP resulted in down regulation of aquaporin-5. The early loss of aquaporin-5 which can be found in different pulmonary fibrosis models does not implicate a specific pathogenetic role during fibrogenesis. PMID:24941004

  13. [Effect of P2X7 receptor knock-out on bone cancer pain in mice].

    PubMed

    Zhao, Xin; Liu, Hui-Zhu; Zhang, Yu-Qiu

    2016-06-25

    Cancer pain is one of the most common symptoms in patients with late stage cancer. Lung, breast and prostate carcinoma are the most common causes of pain from osseous metastasis. P2X7 receptor (P2X7R) is one of the subtypes of ATP-gated purinergic ion channel family, predominately distributed in microglia in the spinal cord. Activation of P2X7Rs in the spinal dorsal horn has been associated with release of proinflammatory cytokines from glial cells, causing increased neuronal excitability and exaggerated nociception. Mounting evidence implies a critical role of P2X7R in inflammatory and neuropathic pain. However, whether P2X7R is involved in cancer pain remains controversial. Here we established a bone cancer pain model by injecting the Lewis lung carcinoma cells into the femur bone marrow cavity of C57BL/6J wild-type mice (C57 WT mice) and P2X7R knockout mice (P2rx7(-/-) mice) to explore the role of P2X7R in bone cancer pain. Following intrafemur carcinoma inoculation, robust mechanical allodynia and thermal hyperalgesia in C57 WT mice were developed on day 7 and 14, respectively, and persisted for at least 28 days in the ipsilateral hindpaw of the affected limb. CatWalk gait analysis showed significant decreases in the print area and stand phase, and a significant increase in swing phase in the ipsilateral hindpaw on day 21 and 28 after carcinoma cells inoculation. Histopathological sections (hematoxylin and eosin stain) showed that the bone marrow of the affected femur was largely replaced by invading tumor cells, and the femur displayed medullary bone loss and bone destruction on day 28 after inoculation. Unexpectedly, no significant changes in bone cancer-induced hypersensitivity of pain behaviors were found in P2rx7(-/-) mice, and the changes of pain-related values in CatWalk gait analysis even occurred earlier in P2rx7(-/-) mice, as compared with C57 WT mice. Together with our previous study in rats that blockade of P2X7R significantly alleviated bone cancer

  14. Double P2X2/P2X3 Purinergic Receptor Knockout Mice Do Not Taste NaCl or the Artificial Sweetener SC45647

    PubMed Central

    Eddy, Meghan C.; Eschle, Benjamin K.; Barrows, Jennell; Hallock, Robert M.; Finger, Thomas E.

    2009-01-01

    The P2X ionotropic purinergic receptors, P2X2 and P2X3, are essential for transmission of taste information from taste buds to the gustatory nerves. Mice lacking both P2X2 and P2X3 purinergic receptors (P2X2/P2X3Dbl−/−) exhibit no taste-evoked activity in the chorda tympani and glossopharyngeal nerves when stimulated with taste stimuli from any of the 5 classical taste quality groups (salt, sweet, sour, bitter, and umami) nor do the mice show taste preferences for sweet or umami, or avoidance of bitter substances (Finger et al. 2005. ATP signaling is crucial for communication from taste buds to gustatory nerves. Science. 310[5753]:1495–1499). Here, we compare the ability of P2X2/P2X3Dbl−/− mice and P2X2/P2X3Dbl+/+ wild-type (WT) mice to detect NaCl in brief-access tests and conditioned aversion paradigms. Brief-access testing with NaCl revealed that whereas WT mice decrease licking at 300 mM and above, the P2X2/P2X3Dbl−/− mice do not show any change in lick rates. In conditioned aversion tests, P2X2/P2X3Dbl−/− mice did not develop a learned aversion to NaCl or the artificial sweetener SC45647, both of which are easily avoided by conditioned WT mice. The inability of P2X2/P2X3Dbl−/− mice to show avoidance of these taste stimuli was not due to an inability to learn the task because both WT and P2X2/P2X3Dbl−/− mice learned to avoid a combination of SC45647 and amyl acetate (an odor cue). These data suggest that P2X2/P2X3Dbl−/− mice are unable to respond to NaCl or SC45647 as taste stimuli, mirroring the lack of gustatory nerve responses to these substances. PMID:19833661

  15. A novel radioligand for the ATP-gated ion channel P2X7: [3H] JNJ-54232334.

    PubMed

    Lord, Brian; Ameriks, Michael K; Wang, Qi; Fourgeaud, Lawrence; Vliegen, Maarten; Verluyten, Willy; Haspeslagh, Pieter; Carruthers, Nicholas I; Lovenberg, Timothy W; Bonaventure, Pascal; Letavic, Michael A; Bhattacharya, Anindya

    2015-10-15

    The ATP-gated ion channel P2X7 has emerged as a potential central nervous system (CNS) drug target based on the hypotheses that pro-inflammatory cytokines such as IL-1β that are released by microglia, may contribute to the etiology of various disorders of the CNS including depression. In this study, we identified two closely related P2X7 antagonists, JNJ-54232334 and JNJ-54140515, and then tritium labeled the former to produce a new radioligand for P2X7. JNJ-54232334 is a high affinity ligand for the rat P2X7 with a pKi of 9.3±0.1. In rat cortical membranes, [3H] JNJ-54232334 reached saturable binding with equilibrium dissociation (Kd) constant of 4.9±1.3 nM. The compound displayed monophasic association and dissociation kinetics with fast on and off rates. In rat brain sections, specific binding of [3H] JNJ-54232334 was markedly improved compared to the previously described P2X7 radioligand, [3H] A-804598. In P2X7 knockout mouse brain sections, [3H] A-804598 bound to non-P2X7 binding sites in contrast to [3H] JNJ-54232334. In rat or wild type mouse brain sections [3H] JNJ-54232334 bound in a more homogenous and region independent manner. The ubiquitous expression of P2X7 receptors was confirmed with immunohistochemistry in rat brain sections. The partial displacement of [3H] A-804598 binding resulted in the underestimation of the level of ex vivo P2X7 occupancy for JNJ-54140515. Higher levels of P2X7 ex vivo occupancy were measured using [3H] JNJ-54232334 due to less non-specific binding. In summary, we describe [3H] JNJ-54232334 as a novel P2X7 radioligand, with improved properties over [3H] A-804598. PMID:26386289

  16. Selective P2X7 receptor antagonists for chronic inflammation and pain

    PubMed Central

    Donnelly-Roberts, Diana; Jarvis, Michael F.

    2008-01-01

    ATP, acting on P2X7 receptors, stimulates changes in intracellular calcium concentrations, maturation, and release of interleukin-1β (IL-1β), and following prolonged agonist exposure, cell death. The functional effects of P2X7 receptor activation facilitate several proinflammatory processes associated with arthritis. Within the nervous system, these proinflammatory processes may also contribute to the development and maintenance of chronic pain. Emerging data from genetic knockout studies have indicated specific roles for P2X7 receptors in inflammatory and neuropathic pain states. The discovery of multiple distinct chemical series of potent and highly selective P2X7 receptor antagonists have enhanced our understanding of P2X7 receptor pharmacology and the diverse array of P2X7 receptor signaling mechanisms. These antagonists have provided mechanistic insight into the role(s) P2X7 receptors play under pathophysiological conditions. In this review, we integrate the recent discoveries of novel P2X7 receptor-selective antagonists with a brief update on P2X7 receptor pharmacology and its therapeutic potential. PMID:18568426

  17. HSP90 Regulation of P2X7 Receptor Function Requires an Intact Cytoplasmic C-Terminus.

    PubMed

    Migita, Keisuke; Ozaki, Taku; Shimoyama, Shuji; Yamada, Junko; Nikaido, Yoshikazu; Furukawa, Tomonori; Shiba, Yuko; Egan, Terrance M; Ueno, Shinya

    2016-08-01

    P2X7 receptors (P2X7Rs) are ATP-gated ion channels that display the unusual property of current facilitation during long applications of agonists. Here we show that facilitation disappears in chimeric P2X7Rs containing the C-terminus of the P2X2 receptor (P2X2R), and in a truncated P2X7R missing the cysteine-rich domain of the C-terminus. The chimeric and truncated receptors also show an apparent decreased permeability to N-methyl-d-glucamine(+) (NMDG(+)). The effects of genetic modification of the C-terminus on NMDG(+) permeability were mimicked by preapplication of the HSP90 antagonist geldanamycin to the wild-type receptor. Further, the geldanamycin decreased the shift in the reversal potential of the ATP-gated current measured under bi-ionic NMDG(+)/Na(+) condition without affecting the ability of the long application of agonist to facilitate current amplitude. Taken together, the results suggest that HSP90 may be essential for stabilization and function of P2X7Rs through an action on the cysteine-rich domain of the cytoplasmic the C-terminus. PMID:27301716

  18. Macrophage activation and polarization modify P2X7 receptor secretome influencing the inflammatory process

    PubMed Central

    de Torre-Minguela, Carlos; Barberà-Cremades, Maria; Gómez, Ana I.; Martín-Sánchez, Fátima; Pelegrín, Pablo

    2016-01-01

    The activation of P2X7 receptor (P2X7R) on M1 polarized macrophages induces the assembly of the NLRP3 inflammasome leading to the release of pro-inflammatory cytokines and the establishment of the inflammatory response. However, P2X7R signaling to the NLRP3 inflammasome is uncoupled on M2 macrophages without changes on receptor activation. In this study, we analyzed P2X7R secretome in wild-type and P2X7R-deficient macrophages polarized either to M1 or M2 and proved that proteins released after P2X7R stimulation goes beyond caspase-1 secretome. The characterization of P2X7R-secretome reveals a new function of this receptor through a fine-tuning of protein release. We found that P2X7R stimulation in macrophages is able to release potent anti-inflammatory proteins, such as Annexin A1, independently of their polarization state suggesting for first time a potential role for P2X7R during resolution of the inflammation and not linked to the release of pro-inflammatory cytokines. These results are of prime importance for the development of therapeutics targeting P2X7R. PMID:26935289

  19. P2X7 receptor antagonist activity of the anti-allergic agent oxatomide.

    PubMed

    Yoshida, Kazuki; Ito, Masaaki; Matsuoka, Isao

    2015-11-15

    Activation of the P2X7 receptor by extracellular ATP is associated with various immune responses including allergic inflammation. Anti-allergic agents, such as H1-antihistamines, are known to inhibit the effects of different chemical mediators such as acetylcholine and platelet-activating factor. Therefore, we hypothesized that some anti-allergic agents might affect P2X7 receptor function. Using N18TG2 and J774 cells, which express functional P2X7 receptors, the effects of several anti-allergic agents on P2X7 receptor function were investigated by monitoring the ATP-induced increase in intracellular Ca(2+) concentrations ([Ca(2+)]i). Among the various agents tested, oxatomide significantly inhibited P2X7 receptor-mediated [Ca(2+)]i elevation in a concentration-dependent manner without affecting the P2Y2 receptor-mediated response in both N18TG2 and J774 cells. Consistently, oxatomide inhibited P2X7 receptor-mediated membrane current and downstream responses such as mitogen-activated protein kinase activation, inflammation-related gene induction, and cell death. In addition, oxatomide inhibited P2X7 receptor-mediated degranulation in mouse bone marrow-derived mast cells. Whole cell patch clamp analyses in HEK293 cells expressing human, mouse, and rat P2X7 receptors revealed that the inhibitory effect of oxatomide on ATP-induced current was most prominent for the human P2X7 receptor and almost non-existent for the rat P2X7 receptor. The potent inhibitory effects of oxatomide on human P2X7 receptor-mediated function were confirmed in RPMI8226 human B cell-like myeloma cells, which endogenously express the P2X7 receptor. Our results demonstrated that the antihistamine oxatomide also acts as a P2X7 receptor antagonist. Future studies should thus evaluate whether P2X7 receptor antagonism contributes to the anti-allergic effects of oxatomide. PMID:26463039

  20. Emerging key roles for P2X receptors in the kidney

    PubMed Central

    Birch, R. E.; Schwiebert, E. M.; Peppiatt-Wildman, C. M.; Wildman, S. S.

    2013-01-01

    P2X ionotropic non-selective cation channels are expressed throughout the kidney and are activated in a paracrine or autocrine manner following the binding of extracellular ATP and related extracellular nucleotides. Whilst there is a wealth of literature describing a regulatory role of P2 receptors (P2R) in the kidney, there are significantly less data on the regulatory role of P2X receptors (P2XR) compared with that described for metabotropic P2Y. Much of the historical literature describing a role for P2XR in the kidney has focused heavily on the role of P2X1R in the autoregulation of renal blood flow. More recently, however, there has been a plethora of manuscripts providing compelling evidence for additional roles for P2XR in both kidney health and disease. This review summarizes the current evidence for the involvement of P2XR in the regulation of renal tubular and vascular function, and highlights the novel data describing their putative roles in regulating physiological and pathophysiological processes in the kidney. PMID:24098285

  1. Emerging key roles for P2X receptors in the kidney.

    PubMed

    Birch, R E; Schwiebert, E M; Peppiatt-Wildman, C M; Wildman, S S

    2013-01-01

    P2X ionotropic non-selective cation channels are expressed throughout the kidney and are activated in a paracrine or autocrine manner following the binding of extracellular ATP and related extracellular nucleotides. Whilst there is a wealth of literature describing a regulatory role of P2 receptors (P2R) in the kidney, there are significantly less data on the regulatory role of P2X receptors (P2XR) compared with that described for metabotropic P2Y. Much of the historical literature describing a role for P2XR in the kidney has focused heavily on the role of P2X1R in the autoregulation of renal blood flow. More recently, however, there has been a plethora of manuscripts providing compelling evidence for additional roles for P2XR in both kidney health and disease. This review summarizes the current evidence for the involvement of P2XR in the regulation of renal tubular and vascular function, and highlights the novel data describing their putative roles in regulating physiological and pathophysiological processes in the kidney. PMID:24098285

  2. Cloning and pharmacological characterization of the dog P2X7 receptor

    PubMed Central

    Roman, S; Cusdin, FS; Fonfria, E; Goodwin, JA; Reeves, J; Lappin, SC; Chambers, L; Walter, DS; Clay, WC; Michel, AD

    2009-01-01

    Background and purpose: Human and rodent P2X7 receptors exhibit differences in their sensitivity to antagonists. In this study we have cloned and characterized the dog P2X7 receptor to determine if its antagonist sensitivity more closely resembles the human or rodent orthologues. Experimental approach: A cDNA encoding the dog P2X7 receptor was isolated from a dog heart cDNA library, expressed in U-2 OS cells using the BacMam viral expression system and characterized in electrophysiological, ethidium accumulation and radioligand binding studies. Native P2X7 receptors were examined by measuring ATP-stimulated interleukin-1β release in dog and human whole blood. Key results: The dog P2X7 receptor was 595 amino acids long and exhibited high homology (>70%) to the human and rodent orthologues although it contained an additional threonine at position 284 and an amino acid deletion at position 538. ATP possessed low millimolar potency at dog P2X7 receptors. 2′-&3′-O-(4benzoylbenzoyl) ATP had slightly higher potency but was a partial agonist. Dog P2X7 receptors possessed relatively high affinity for a number of selective antagonists of the human P2X7 receptor although there were some differences in potency between the species. Compound affinities in human and dog blood exhibited a similar rank order of potency as observed in studies on the recombinant receptor although absolute potency was considerably lower. Conclusions and implications: Dog recombinant and native P2X7 receptors display a number of pharmacological similarities to the human P2X7 receptor. Thus, dog may be a suitable species for assessing target-related toxicity of antagonists intended for evaluation in the clinic. PMID:19814727

  3. Extracellular ATP protects against sepsis through macrophage P2X7 purinergic receptors by enhancing intracellular bacterial killing.

    PubMed

    Csóka, Balázs; Németh, Zoltán H; Törő, Gábor; Idzko, Marco; Zech, Andreas; Koscsó, Balázs; Spolarics, Zoltán; Antonioli, Luca; Cseri, Karolina; Erdélyi, Katalin; Pacher, Pál; Haskó, György

    2015-09-01

    Extracellular ATP binds to and signals through P2X7 receptors (P2X7Rs) to modulate immune function in both inflammasome-dependent and -independent manners. In this study, P2X7(-/-) mice, the pharmacological agonists ATP-magnesium salt (Mg-ATP; 100 mg/kg, EC50 ≈ 1.32 mM) and benzoylbenzoyl-ATP (Bz-ATP; 10 mg/kg, EC50 ≈ 285 μM), and antagonist oxidized ATP (oxi-ATP; 40 mg/kg, IC50 ≈ 100 μM) were used to show that P2X7R activation is crucial for the control of mortality, bacterial dissemination, and inflammation in cecal ligation and puncture-induced polymicrobial sepsis in mice. Our results with P2X7(-/-) bone marrow chimeric mice, adoptive transfer of peritoneal macrophages, and myeloid-specific P2X7(-/-) mice indicate that P2X7R signaling on macrophages is essential for the protective effect of P2X7Rs. P2X7R signaling protects through enhancing bacterial killing by macrophages, which is independent of the inflammasome. By using the connexin (Cx) channel inhibitor Gap27 (0.1 mg/kg, IC50 ≈ 0.25 μM) and pannexin channel inhibitor probenecid (10 mg/kg, IC50 ≈ 11.7 μM), we showed that ATP release through Cx is important for inhibiting inflammation and bacterial burden. In summary, targeting P2X7Rs provides a new opportunity for harnessing an endogenous protective immune mechanism in the treatment of sepsis. PMID:26060214

  4. Knocking out P2X receptors reduces transmitter secretion in taste buds

    PubMed Central

    Huang, Yijen A.; Stone, Leslie M.; Pereira, Elizabeth; Yang, Ruibiao; Kinnamon, John C.; Dvoryanchikov, Gennady; Chaudhari, Nirupa; Finger, Thomas E.; Kinnamon, Sue C.; Roper, Stephen D.

    2011-01-01

    In response to gustatory stimulation, taste bud cells release a transmitter, ATP, that activates P2X2 and P2X3 receptors on gustatory afferent fibers. Taste behavior and gustatory neural responses are largely abolished in mice lacking P2X2 and P2X3 receptors (P2X2 and P2X3 double knockout, or “DKO” mice). The assumption has been that eliminating P2X2 and P2X3 receptors only removes postsynaptic targets but that transmitter secretion in mice is normal. Using functional imaging, ATP biosensor cells, and a cell-free assay for ATP, we tested this assumption. Surprisingly, although gustatory stimulation mobilizes Ca2+ in taste Receptor (Type II) cells from DKO mice, as from wild type (WT) mice, taste cells from DKO mice fail to release ATP when stimulated with tastants. ATP release could be elicited by depolarizing DKO Receptor cells with KCl, suggesting that ATP-release machinery remains functional in DKO taste buds. To explore the difference in ATP release across genotypes, we employed reverse transcriptase (RT)-PCR, immunostaining, and histochemistry for key proteins underlying ATP secretion and degradation: Pannexin1, TRPM5, and NTPDase2 (ecto-ATPase) are indistinguishable between WT and DKO mice. The ultrastructure of contacts between taste cells and nerve fibers is also normal in the DKO mice. Finally, quantitative RT-PCR show that P2X4 and P2X7, potential modulators of ATP secretion, are similarly expressed in taste buds in WT and DKO taste buds. Importantly, we find that P2X2 is expressed in WT taste buds and appears to function as an autocrine, positive feedback signal to amplify taste-evoked ATP secretion. PMID:21940456

  5. P2X3 receptors and sensitization of autonomic reflexes.

    PubMed

    Ford, Anthony P; Undem, Bradley J; Birder, Lori A; Grundy, David; Pijacka, Wioletta; Paton, Julian F R

    2015-09-01

    A great deal of basic and applied physiology and pharmacology in sensory and autonomic neuroscience has teased apart mechanisms that drive normal perception of mechanical, thermal and chemical signals and convey them to CNS, the distinction of fiber types and receptors and channels that mediate them, and how they may become dysfunctional or maladaptive in disease. Likewise, regulation of efferent autonomic traffic to control organ reflexes has been well studied. In both afferent and efferent limbs, a wide array of potential therapeutic mechanisms has surfaced, some of which have progressed into clinic, if not full regrastration. One conversation that has been less well progressed relates to how the afferent limb and its sensitization shapes the efferent outputs, and where modulation may offer new therapeutic avenues, especially for poorly addressed and common signs and symptoms of disease. Therapeutics for CV disease (HF, hypertension), respiratory disease (asthma, COPD), urological disease (OAB), GI disease (IBS), and inter alia, have largely focused on the efferent control of effector cells to modulate movement, contraction and secretion; medicinal needs remain with limits to efficacy, AEs and treatment resistance being common. We now must turn, in the quest for improved therapeutics, to understand how sensation from these organs becomes maladapted and sensitized in disease, and what opportunities may arise for improved therapeutics given the abundance of targets, many pharmacologically untapped, on the afferent side. One might look at the treatment resistant hypertension and the emerging benefit of renal denervation; or urinary bladder overactivity / neurogenic bladder and the emergence of neuromodulation, capsaicin instillation or botox injections to attenuate sensitized reflexes, as examples of merely the start of such progress. This review examines this topic more deeply, as applies to four major organ systems all sharing a great need from unsatisfied

  6. Modulating P2X7 Receptor Signaling during Rheumatoid Arthritis: New Therapeutic Approaches for Bisphosphonates.

    PubMed

    Baroja-Mazo, Alberto; Pelegrín, Pablo

    2012-01-01

    P2X7 receptor-mediated purinergic signaling is a well-known mechanism involved in bone remodeling. The P2X7 receptor has been implicated in the pathophysiology of various bone and cartilage diseases, including rheumatoid arthritis (RA), a widespread and complex chronic inflammatory disorder. The P2X7 receptor induces the release into the synovial fluid of the proinflammatory factors (e.g., interleukin-1β, prostaglandins, and proteases) responsible for the clinical symptoms of RA. Thus, the P2X7 receptor is emerging as a novel anti-inflammatory therapeutic target, and various selective P2X7 receptor antagonists are under clinical trials. Extracellular ATP signaling acting through the P2X7 receptor is a complex and dynamic scenario, which varies over the course of inflammation. This signaling is partially modulated by the activity of ectonucleotidases, which degrade extracellular ATP to generate other active molecules such as adenosine or pyrophosphates. Recent evidence suggests differential extracellular metabolism of ATP during the resolution of inflammation to generate pyrophosphates. Extracellular pyrophosphate dampens proinflammatory signaling by promoting alternative macrophage activation. Our paper shows that bisphosphonates are metabolically stable pyrophosphate analogues that are able to mimic the anti-inflammatory function of pyrophosphates. Bisphosphonates are arising per se as promising anti-inflammatory drugs to treat RA, and this therapy could be improved when administrated in combination with P2X7 receptor antagonists. PMID:22830074

  7. P2X7 receptor as predictor gene for glioma radiosensitivity and median survival.

    PubMed

    Gehring, Marina P; Kipper, Franciele; Nicoletti, Natália F; Sperotto, Nathalia D; Zanin, Rafael; Tamajusuku, Alessandra S K; Flores, Debora G; Meurer, Luise; Roesler, Rafael; Filho, Aroldo B; Lenz, Guido; Campos, Maria M; Morrone, Fernanda B

    2015-11-01

    Glioblastoma multiforme (GBM) is considered the most lethal intracranial tumor and the median survival time is approximately 14 months. Although some glioma cells present radioresistance, radiotherapy has been the mainstay of therapy for patients with malignant glioma. The activation of P2X7 receptor (P2X7R) is responsible for ATP-induced death in various cell types. In this study, we analyzed the importance of ATP-P2X7R pathway in the radiotherapy response P2X7R silenced cell lines, in vivo and human tumor samples. Both glioma cell lines used in this study present a functional P2X7R and the P2X7R silencing reduced P2X7R pore activity by ethidium bromide uptake. Gamma radiation (2Gy) treatment reduced cell number in a P2X7R-dependent way, since both P2X7R antagonist and P2X7R silencing blocked the cell cytotoxicity caused by irradiation after 24h. The activation of P2X7R is time-dependent, as EtBr uptake significantly increased after 24h of irradiation. The radiotherapy plus ATP incubation significantly increased annexin V incorporation, compared with radiotherapy alone, suggesting that ATP acts synergistically with radiotherapy. Of note, GL261 P2X7R silenced-bearing mice failed in respond to radiotherapy (8Gy) and GL261 WT-bearing mice, that constitutively express P2X7R, presented a significant reduction in tumor volume after radiotherapy, showing in vivo that functional P2X7R expression is essential for an efficient radiotherapy response in gliomas. We also showed that a high P2X7R expression is a good prognostic factor for glioma radiosensitivity and survival probability in humans. Our data revealed the relevance of P2X7R expression in glioma cells to a successful radiotherapy response, and shed new light on this receptor as a useful predictor of the sensitivity of cancer patients to radiotherapy and median survival. PMID:26358881

  8. Accelerated tumor progression in mice lacking the ATP receptor P2X7.

    PubMed

    Adinolfi, Elena; Capece, Marina; Franceschini, Alessia; Falzoni, Simonetta; Giuliani, Anna L; Rotondo, Alessandra; Sarti, Alba C; Bonora, Massimo; Syberg, Susanne; Corigliano, Domenica; Pinton, Paolo; Jorgensen, Niklas R; Abelli, Luigi; Emionite, Laura; Raffaghello, Lizzia; Pistoia, Vito; Di Virgilio, Francesco

    2015-02-15

    The ATP receptor P2X7 (P2X7R or P2RX7) has a key role in inflammation and immunity, but its possible roles in cancer are not firmly established. In the present study, we investigated the effect of host genetic deletion of P2X7R in the mouse on the growth of B16 melanoma or CT26 colon carcinoma cells. Tumor size and metastatic dissemination were assessed by in vivo calliper and luciferase luminescence emission measurements along with postmortem examination. In P2X7R-deficient mice, tumor growth and metastatic spreading were accelerated strongly, compared with wild-type (wt) mice. Intratumoral IL-1β and VEGF release were drastically reduced, and inflammatory cell infiltration was abrogated nearly completely. Similarly, tumor growth was also greatly accelerated in wt chimeric mice implanted with P2X7R-deficient bone marrow cells, defining hematopoietic cells as a sufficient site of P2X7R action. Finally, dendritic cells from P2X7R-deficient mice were unresponsive to stimulation with tumor cells, and chemotaxis of P2X7R-less cells was impaired. Overall, our results showed that host P2X7R expression was critical to support an antitumor immune response, and to restrict tumor growth and metastatic diffusion. PMID:25542861

  9. Molecular Mechanisms of Cross-inhibition Between Nicotinic Acetylcholine Receptors and P2X Receptors in Myenteric Neurons and HEK-293 cells

    PubMed Central

    Decker, Dima A.; Galligan, James J.

    2010-01-01

    Background P2X2 and nicotinic acetylcholine receptors (nAChRs) mediate fast synaptic excitation in the enteric nervous system. P2X receptors and nAChRs are functionally linked. This study examined the mechanisms responsible for interactions between P2X2 and α3β4subunit-containing nAChRs. Methods The function of P2X2 and α3β4 nAChRs expressed by HEK-293 cells and guinea pig ileum myenteric neurons in culture was studied using whole-cell patch clamp techniques. Results In HEK-293 cells expressing α3β4 nAChRs and P2X2 receptors, co-application of ATP and ACh caused inward currents that were 56 ± 7% of the current that should occur if these channels functioned independently (P < 0.05, n = 9); we call this interaction cross-inhibition. Cross-inhibition did not occur in HEK-293 cells expressing α3β4 nAChRs and a C-terminal tail truncated P2X2 receptor (P2X2TR)(P >0.05, n = 8). Intracellular application of the C-terminal tail of the P2X2 receptor blocked nAChR-P2X receptor cross-inhibition in HEK-293 cells and myenteric neurons. In the absence of ATP, P2X2 receptors constitutively inhibited nAChR currents in HEK-293 cells expressing both receptors. Constitutive inhibition did not occur in HEK-293 cells expressing α3β4 nAChRs transfected with P2X2TR. Currents caused by low (≤30 μM), but not high (≥100 μM) concentrations of ATP in cells expressing P2X2 receptors were inhibited by co-expression with α3β4 nAChRs. Conclusions The C-terminal tail of P2X2 receptors mediates cross-inhibition between α3β4 nAChR-P2X2 receptors. The closed state of P2X2 receptors and nAChRs can also cause cross inhibition. These interactions may modulate transmission at enteric synapses that use ATP and acetylcholine as co-transmitters. PMID:20426799

  10. Medicinal chemistry of adenosine, P2Y and P2X receptors.

    PubMed

    Jacobson, Kenneth A; Müller, Christa E

    2016-05-01

    Pharmacological tool compounds are now available to define action at the adenosine (ARs), P2Y and P2X receptors. We present a selection of the most commonly used agents to study purines in the nervous system. Some of these compounds, including A1 and A3 AR agonists, P2Y1R and P2Y12R antagonists, and P2X3, P2X4 and P2X7 antagonists, are potentially of clinical use in treatment of disorders of the nervous system, such as chronic pain, neurodegeneration and brain injury. Agonists of the A2AAR and P2Y2R are already used clinically, P2Y12R antagonists are widely used antithrombotics and an antagonist of the A2AAR is approved in Japan for treating Parkinson's disease. The selectivity defined for some of the previously introduced compounds has been revised with updated pharmacological characterization, for example, various AR agonists and antagonists were deemed A1AR or A3AR selective based on human data, but species differences indicated a reduction in selectivity ratios in other species. Also, many of the P2R ligands still lack bioavailability due to charged groups or hydrolytic (either enzymatic or chemical) instability. X-ray crystallographic structures of AR and P2YRs have shifted the mode of ligand discovery to structure-based approaches rather than previous empirical approaches. The X-ray structures can be utilized either for in silico screening of chemically diverse libraries for the discovery of novel ligands or for enhancement of the properties of known ligands by chemical modification. Although X-ray structures of the zebrafish P2X4R have been reported, there is scant structural information about ligand recognition in these trimeric ion channels. In summary, there are definitive, selective agonists and antagonists for all of the ARs and some of the P2YRs; while the pharmacochemistry of P2XRs is still in nascent stages. The therapeutic potential of selectively modulating these receptors is continuing to gain interest in such fields as cancer, inflammation, pain

  11. P2X receptors and their roles in astroglia in the central and peripheral nervous system.

    PubMed

    Illes, Peter; Verkhratsky, Alexei; Burnstock, Geoffrey; Franke, Heike

    2012-10-01

    Astrocytes are a class of neural cells that control homeostasis at all levels of the central and peripheral nervous system. There is a bidirectional neuron-glia interaction via a number of extracellular signaling molecules, glutamate and ATP being the most widespread. ATP activates ionotropic P2X and metabotropic P2Y receptors, which operate in both neurons and astrocytes. Morphological, biochemical, and functional evidence indicates the expression of astroglial P2X(1/5) heteromeric and P2X(7) homomeric receptors, which mediate physiological and pathophysiological responses. Activation of P2X(1/5) receptors triggers rapid increase of intracellular Na(+) that initiates immediate cellular reactions, such as the depression of the glutamate transporter to keep high glutamate concentrations in the synaptic cleft, the activation of the local lactate shuttle to supply energy substrate to pre- and postsynaptic neuronal structures, and the reversal of the Na(+)/Ca(2+) exchange resulting in additional Ca(2+) entry. The consequences of P2X(7) receptor activation are mostly but not exclusively mediated by the entry of Ca(2+) and result in reorganization of the cytoskeleton, inflammation, apoptosis/necrosis, and proliferation, usually at a prolonged time scale. Thus, astroglia detect by P2X(1/5) and P2X(7) receptors both physiological concentrations of ATP secreted from presynaptic nerve terminals and also much higher concentrations of ATP attained under pathological conditions. PMID:22013151

  12. Purinergic signaling mediated by P2X7 receptors controls myelination in sciatic nerves.

    PubMed

    Faroni, A; Smith, R J P; Procacci, P; Castelnovo, L F; Puccianti, E; Reid, A J; Magnaghi, V; Verkhratsky, A

    2014-10-01

    Adenosine-5'-triphosphate, the physiological ligand of P2X receptors, is an important factor in peripheral nerve development. P2X7 receptor is expressed in Schwann cells (SCs), but the specific effects of P2X7 purinergic signaling on peripheral nerve development, myelination, and function are largely unknown. In this study, sciatic nerves from P2X7 knockout mice were analyzed for altered expression of myelin-associated proteins and for alterations in nerve morphology. Immunohistochemical analyses revealed that, in the wild-type peripheral nerves, the P2X7 receptor was localized mainly in myelinating SCs, with only a few immunopositive nonmyelinating SCs. Complete absence of P2X7 receptor protein was confirmed in the sciatic nerves of the knockout mice by Western blot and immunohistochemistry. Western blot analysis revealed that expression levels of the myelin proteins protein zero and myelin-associated glycoprotein are reduced in P2X7 knockout nerves. In accordance with the molecular results, transmission electron microscopy analyses revealed that P2X7 knockout nerves possess significantly more unmyelinated axons, contained in a higher number of Remak bundles. The myelinating/nonmyelinating SC ratio was also decreased in knockout mice, and we found a significantly increased number of irregular fibers compared with control nerves. Nevertheless, the myelin thickness in the knockout was unaltered, suggesting a stronger role for P2X7 in determining SC maturation than in myelin formation. In conclusion, we present morphological and molecular evidence of the importance of P2X7 signaling in peripheral nerve maturation and in determining SC commitment to a myelinating phenotype. PMID:24903685

  13. The ATP Receptors P2X7 and P2X4 Modulate High Glucose and Palmitate-Induced Inflammatory Responses in Endothelial Cells

    PubMed Central

    Sathanoori, Ramasri; Swärd, Karl; Olde, Björn; Erlinge, David

    2015-01-01

    Endothelial cells lining the blood vessels are principal players in vascular inflammatory responses. Dysregulation of endothelial cell function caused by hyperglycemia, dyslipidemia, and hyperinsulinemia often result in impaired vasoregulation, oxidative stress, inflammation, and altered barrier function. Various stressors including high glucose stimulate the release of nucleotides thus initiating signaling via purinergic receptors. However, purinergic modulation of inflammatory responses in endothelial cells caused by high glucose and palmitate remains unclear. In the present study, we investigated whether the effect of high glucose and palmitate is mediated by P2X7 and P2X4 and if they play a role in endothelial cell dysfunction. Transcript and protein levels of inflammatory genes as well as reactive oxygen species production, endothelial-leukocyte adhesion, and cell permeability were investigated in human umbilical vein endothelial cells exposed to high glucose and palmitate. We report high glucose and palmitate to increase levels of extracellular ATP, expression of P2X7 and P2X4, and inflammatory markers. Both P2X7 and P2X4 antagonists inhibited high glucose and palmitate-induced interleukin-6 levels with the former having a significant effect on interleukin-8 and cyclooxygenase-2. The effect of the antagonists was confirmed with siRNA knockdown of the receptors. In addition, P2X7 mediated both high glucose and palmitate-induced increase in reactive oxygen species levels and decrease in endothelial nitric oxide synthase. Blocking P2X7 inhibited high glucose and palmitate-induced expression of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 as well as leukocyte-endothelial cell adhesion. Interestingly, high glucose and palmitate enhanced endothelial cell permeability that was dependent on both P2X7 and P2X4. Furthermore, antagonizing the P2X7 inhibited high glucose and palmitate-mediated activation of p38-mitogen activated protein kinase

  14. Crystal structure of the ATP-gated P2X[subscript 4] ion channel in the closed state

    SciTech Connect

    Kawate, Toshimitsu; Michel, Jennifer Carlisle; Birdsong, William T.; Gouaux, Eric

    2009-08-13

    P2X receptors are cation-selective ion channels gated by extracellular ATP, and are implicated in diverse physiological processes, from synaptic transmission to inflammation to the sensing of taste and pain. Because P2X receptors are not related to other ion channel proteins of known structure, there is at present no molecular foundation for mechanisms of ligand-gating, allosteric modulation and ion permeation. Here we present crystal structures of the zebrafish P2X{sub 4} receptor in its closed, resting state. The chalice-shaped, trimeric receptor is knit together by subunit-subunit contacts implicated in ion channel gating and receptor assembly. Extracellular domains, rich in {beta}-strands, have large acidic patches that may attract cations, through fenestrations, to vestibules near the ion channel. In the transmembrane pore, the 'gate' is defined by an {approx}8 {angstrom} slab of protein. We define the location of three non-canonical, intersubunit ATP-binding sites, and suggest that ATP binding promotes subunit rearrangement and ion channel opening.

  15. P2X7 receptors mediate deleterious renal epithelial-fibroblast cross talk.

    PubMed

    Ponnusamy, Murugavel; Ma, Li; Gong, Rujun; Pang, Maoyin; Chin, Y Eugene; Zhuang, Shougang

    2011-01-01

    Peritubular fibroblasts in the kidney are the major erythropoietin-producing cells and also contribute to renal repair following acute kidney injury (AKI). Although few fibroblasts were observed in the interstitium adjacent to damaged tubular epithelium in the early phase of AKI, the underlying mechanism by which their numbers were reduced remains unknown. In this study, we tested the hypothesis that damaged renal epithelial cells directly induce renal interstitial fibroblast death by releasing intracellular ATP and activating purinergic signaling. Exposure of a cultured rat renal interstitial fibroblast cell line (NRK-49F) to necrotic renal proximal tubular cells (RPTC) lysate or supernatant induced NRK-49F cell death by apoptosis and necrosis. Depletion of ATP with apyrase or inhibition of the P2X purinergic receptor with pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid blocked the deleterious effect of necrotic RPTC supernatant. The P2X7 receptor, an ATP-sensitive purinergic receptor, was not detected in cultured NRK-49F cells but was inducible by necrotic RPTC supernatant. Treatment with A438079, a highly selective P2X7 receptor inhibitor, or knockdown of the P2X7 receptor with small interference RNA diminished renal fibroblast death induced by necrotic RPTC supernatant. Conversely, overexpression of the P2X7 receptor potentiated this response. Collectively, these findings provide strong evidence that damaged renal epithelial cells can directly induce the death of renal interstitial fibroblasts by ATP activation of the P2X7 receptor. PMID:20861083

  16. Physiological role for P2X1 receptors in renal microvascular autoregulatory behavior

    PubMed Central

    Inscho, Edward W.; Cook, Anthony K.; Imig, John D.; Vial, Catherine; Evans, Richard J.

    2003-01-01

    This study tests the hypothesis that P2X1 receptors mediate pressure-induced afferent arteriolar autoregulatory responses. Afferent arterioles from rats and P2X1 KO mice were examined using the juxtamedullary nephron technique. Arteriolar diameter was measured in response to step increases in renal perfusion pressure (RPP). Autoregulatory adjustments in diameter were measured before and during P2X receptor blockade with NF279 or A1 receptor blockade with 1,3-dipropyl-8-cyclopentylxanthine (DPCPX). Acute papillectomy or furosemide perfusion was performed to interrupt distal tubular fluid flow past the macula densa, thus minimizing tubuloglomerular feedback–dependent influences on afferent arteriolar function. Under control conditions, arteriolar diameter decreased by 17% and 29% at RPP of 130 and 160 mmHg, respectively. Blockade of P2X1 receptors with NF279 blocked pressure-mediated vasoconstriction, reflecting an attenuated autoregulatory response. The A1 receptor blocker DPCPX did not alter autoregulatory behavior or the response to ATP. Deletion of P2X1 receptors in KO mice significantly blunted autoregulatory responses induced by an increase in RPP, and this response was not further impaired by papillectomy or furosemide. WT control mice exhibited typical RPP-dependent vasoconstriction that was significantly attenuated by papillectomy. These data provide compelling new evidence indicating that tubuloglomerular feedback signals are coupled to autoregulatory preglomerular vasoconstriction through ATP-mediated activation of P2X1 receptors. PMID:14679185

  17. Caveolin-1 regulates P2X7 receptor signaling in osteoblasts

    PubMed Central

    Gangadharan, Vimal; Nohe, Anja; Caplan, Jeffrey; Czymmek, Kirk

    2014-01-01

    The synthesis of new bone in response to a novel applied mechanical load requires a complex series of cellular signaling events in osteoblasts and osteocytes. The activation of the purinergic receptor P2X7R is central to this mechanotransduction signaling cascade. Recently, P2X7R have been found to be associated with caveolae, a subset of lipid microdomains found in several cell types. Deletion of caveolin-1 (CAV1), the primary protein constituent of caveolae in osteoblasts, results in increased bone mass, leading us to hypothesize that the P2X7R is scaffolded to caveolae in osteoblasts. Thus, upon activation of the P2X7R, we postulate that caveolae are endocytosed, thereby modulating the downstream signal. Sucrose gradient fractionation of MC3T3-E1 preosteoblasts showed that CAV1 was translocated to the denser cytosolic fractions upon stimulation with ATP. Both ATP and the more specific P2X7R agonist 2′(3′)-O-(4-benzoylbenzoyl)ATP (BzATP) induced endocytosis of CAV1, which was inhibited when MC3T3-E1 cells were pretreated with the specific P2X7R antagonist A-839977. The P2X7R cofractionated with CAV1, but, using superresolution structured illumination microscopy, we found only a subpopulation of P2X7R in these lipid microdomains on the membrane of MC3T3-E1 cells. Suppression of CAV1 enhanced the intracellular Ca2+ response to BzATP, suggesting that caveolae regulate P2X7R signaling. This proposed mechanism is supported by increased mineralization in CAV1 knockdown MC3T3-E1 cells treated with BzATP. These data suggest that caveolae regulate P2X7R signaling upon activation by undergoing endocytosis and potentially carrying with it other signaling proteins, hence controlling the spatiotemporal signaling of P2X7R in osteoblasts. PMID:25318104

  18. Ivermectin Antagonizes Ethanol Inhibition in Purinergic P2X4 Receptors

    PubMed Central

    Popova, Maya; Perkins, Daya; Trudell, James R.; Alkana, Ronald L.; Davies, Daryl L.

    2010-01-01

    ATP-gated purinergic P2X4 receptors (P2X4Rs) are expressed in the central nervous system and are sensitive to ethanol at intoxicating concentrations. P2XRs are trimeric; each subunit consists of two transmembrane (TM) α-helical segments, a large extracellular domain, and intracellular amino and carboxyl terminals. Recent work indicates that position 336 (Met336) in the TM2 segment is critical for ethanol modulation of P2X4Rs. The anthelmintic medication ivermectin (IVM) positively modulates P2X4Rs and is believed to act in the same region as ethanol. The present study tested the hypothesis that IVM can antagonize ethanol action. We investigated IVM and ethanol effects in wild-type and mutant P2X4Rs expressed in Xenopus oocytes by using a two-electrode voltage clamp. IVM antagonized ethanol-induced inhibition of P2X4Rs in a concentration-dependent manner. The size and charge of substitutions at position 336 affected P2X4R sensitivity to both ethanol and IVM. The first molecular model of the rat P2X4R, built onto the X-ray crystal structure of zebrafish P2X4R, revealed a pocket formed by Asp331, Met336, Trp46, and Trp50 that may play a role in the actions of ethanol and IVM. These findings provide the first evidence for IVM antagonism of ethanol effects in P2X4Rs and suggest that the antagonism results from the ability of IVM to interfere with ethanol action on the putative pocket at or near position 336. Taken with the building evidence supporting a role for P2X4Rs in ethanol intake, the present findings suggest that the newly identified alcohol pocket is a potential site for development of medication for alcohol use disorders. PMID:20543096

  19. Saffron reduces ATP-induced retinal cytotoxicity by targeting P2X7 receptors.

    PubMed

    Corso, Lucia; Cavallero, Anna; Baroni, Debora; Garbati, Patrizia; Prestipino, Gianfranco; Bisti, Silvia; Nobile, Mario; Picco, Cristiana

    2016-03-01

    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. PMID:26739703

  20. Pharmacological and molecular characterization of P2X receptors in rat pelvic ganglion neurons

    PubMed Central

    Zhong, Yu; Dunn, Philip M; Xiang, Zhenghua; Bo, Xuenong; Burnstock, Geoffrey

    1998-01-01

    The presence and characteristics of P2X receptors on neurons of the rat major pelvic ganglia (MPG) have been studied using whole cell voltage-clamp, in situ hybridization and immunohistochemistry.Rapid application of ATP (100 μM) to isolated rat MPG neurons induced moderately large inward currents (0.33–5.3 nA) in 39% of cells (108/277). The response to ATP occurred very rapidly, with an increase in membrane conductance, and desensitized slowly.The concentration-response curve for ATP yielded an EC50 of 58.9 μM. The agonist profile was ATP⩾2MeSATP=ATPγS>BzATP, while α,β-MeATP, β,γ-MeATP, UTP and ADP were all inactive at concentrations up to 100 μM.The response to ATP was antagonized by suramin (pA2=5.6), reactive blue-2 (IC50=0.7 μM) and pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS).Lowering the pH from 7.4 to 6.8 produced a marked potentiation (to 339% of control) of the responses to ATP (30 μM), while raising the pH to 8.0 attenuated the responses (to 20% of control). The EC50s for ATP were 28.8, 58.9 and 264 μM at pH 6.8, 7.4 and 8.0, respectively.Co-application of ATP with Zn2+ produced a marked enhancement of the responses to ATP, with an EC50 of 9.55 μM. In the presence of Zn2+ (30 μM), the EC50 for ATP was decreased to 4.57 μM.In situ hybridization revealed that the P2X receptor transcripts levels in rat MPG neurons are P2X2>P2X4>P2X1, P2X3, P2X5 and P2X6. The immunohistochemical staining revealed a small number of neurons with strong P2X2 immunoreactivity.In conclusion, our results indicate that there are P2X receptors present on MPG neurons. The pharmacological characteristics of these receptors, the in situ hybridization and immunohistochemical evidence are consistent with them being of the P2X2 subtype, or heteromultimers, with P2X2 being the dominant component. PMID:9831914

  1. Anoctamin 6 mediates effects essential for innate immunity downstream of P2X7 receptors in macrophages

    NASA Astrophysics Data System (ADS)

    Ousingsawat, Jiraporn; Wanitchakool, Podchanart; Kmit, Arthur; Romao, Ana M.; Jantarajit, Walailak; Schreiber, Rainer; Kunzelmann, Karl

    2015-02-01

    Purinergic P2X7 receptors (P2X7R) are fundamental to innate immune response. In macrophages, transient stimulation of P2X7R activates several transport mechanisms and induces the scrambling of phospholipids with subsequent membrane blebbing and apoptosis. These processes support phagocytosis and subsequent killing of phagocytosed bacteria. Here we demonstrate that the stimulation of P2X7 receptors activates anoctamin 6 (ANO6, TMEM16F), a protein that functions as Ca2+ dependent phospholipid scramblase and Ca2+-activated Cl- channel. Inhibition or knockdown of ANO6 attenuates ATP-induced cell shrinkage, cell migration and phospholipid scrambling. In mouse macrophages, Ano6 produces large ion currents by stimulation of P2X7 receptors and contributes to ATP-induced membrane blebbing and apoptosis, which is largely reduced in macrophages from Ano6-/- mice. ANO6 supports bacterial phagocytosis and killing by mouse and human THP-1 macrophages. Our data demonstrate that anoctamin 6 is an essential component of the immune defense by macrophages.

  2. The penultimate arginine of the carboxy terminus determines slow desensitisation in a P2X receptor from the cattle tick Boophilus microplus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    P2X ion channels have been functionally characterised from a range of eukaryotes. Whilst these receptors can be broadly classified into fast and slow desensitising, the molecular mechanisms underlying current desensitisation are not fully understood. Here we describe the characterisation of a P2X ch...

  3. N-Alkyl-Substituted Isatins Enhance P2X7 Receptor-Induced Interleukin-1β Release from Murine Macrophages

    PubMed Central

    2016-01-01

    Extracellular adenosine 5′-triphosphate (ATP) activates the P2X7 receptor channel to induce the rapid release of the proinflammatory cytokine, interleukin- (IL-) 1β, from macrophages. Microtubule rearrangements are thought to be involved in this process. Some isatin derivatives alter microtubules and display anticancer activities. The current study investigated the effect of isatin and seven structurally diverse isatin derivatives on P2X7-mediated IL-1β release from murine J774 macrophages. ATP-induced IL-1β and lactate dehydrogenase (LDH) release were assessed by specific colorimetric assays. P2X7 activity was determined by flow cytometric measurements of ATP-induced cation dye uptake. Cytotoxicity of isatin derivatives was determined using a tetrazolium-based colorimetric assay. ATP caused rapid IL-1β release in a concentration-dependent manner, and this process was completely impaired by the P2X7 antagonist, AZ10606120. In contrast, 5,7-dibromo-N-(p-methoxybenzyl)isatin (NAI) and 3-{4-[5,7-dibromo-1-(4-methoxybenzyl)-2-oxoindolin-3-ylidenamino]phenyl}propanoic acid (NAI-imine) enhanced P2X7-induced IL-1β release by twofold compared to that of isatin and the parent molecule, 5,7-dibromoisatin. NAI and NAI-imine had minimal effect on P2X7-induced dye uptake and LDH release. In contrast, 24-hour incubation with NAI and NAI-imine (in the absence of exogenous ATP) induced macrophage death in a concentration-dependent manner. In conclusion, this study demonstrates that N-alkyl-substituted isatins enhance P2X7 receptor-induced IL-1β release from murine macrophages. Thus, in addition to direct anticancer effects, these compounds may also impact inflammatory and immune cells within the tumor microenvironment. PMID:27524862

  4. P2X7 receptor predicts postoperative cancer-specific survival of patients with clear-cell renal cell carcinoma.

    PubMed

    Liu, Zheng; Liu, Yidong; Xu, Le; An, Huimin; Chang, Yuan; Yang, Yuanfeng; Zhang, Weijuan; Xu, Jiejie

    2015-09-01

    The P2X7 receptor, an ATP-gated plasma membrane ion channel, is involved in inflammation, apoptosis and cell proliferation, and thereby plays a crucial role during oncogenic transformation in various malignancies. This study aims to evaluate the impact of P2X7 receptor expression on postoperative cancer-specific survival of patients with clear-cell renal cell carcinoma (ccRCC). A total of 273 patients with ccRCC undergoing nephrectomy at a single institution were retrospectively enrolled in this study, among which 86 patients died of this disease and six patients died of other causes. Clinicopathologic features and cancer-specific survival (CSS) were recorded. P2X7 expression was assessed by immunohistochemistry in clinical specimens. Kaplan-Meier method with log rank test was performed to compare survival curves. Cox regression models were used to evaluate the prognostic values of variables on CSS. Concordance index was calculated to assess prognostic accuracy of prognostic models. Median follow-up period was 90 months (range, 11-120 months). Intratumoral P2X7 expression was significantly lower than peritumoral tissues (P < 0.001). Moreover, high intratumoral P2X7 expression, which was significantly associated with shorten CSS (P < 0.001), high TNM stage (P = 0.038), Fuhrman grade (P = 0.035), SSIGN (stage, size, grade, and necrosis) score (P = 0.021) and University of California Integrated Staging System (UISS) score (P = 0.007), was indicated to be an independent prognostic factor for CSS (hazard ratio [HR], 1.693; P = 0.034). The prognostic accuracy of TNM stage, UISS and SSIGN scoring models was improved when intratumoral P2X7 expression was added. Intratumoral P2X7 expression is a potential independent adverse prognostic indicator for postoperative CSS of patients with ccRCC. PMID:26179886

  5. N-Alkyl-Substituted Isatins Enhance P2X7 Receptor-Induced Interleukin-1β Release from Murine Macrophages.

    PubMed

    Sluyter, Ronald; Vine, Kara L

    2016-01-01

    Extracellular adenosine 5'-triphosphate (ATP) activates the P2X7 receptor channel to induce the rapid release of the proinflammatory cytokine, interleukin- (IL-) 1β, from macrophages. Microtubule rearrangements are thought to be involved in this process. Some isatin derivatives alter microtubules and display anticancer activities. The current study investigated the effect of isatin and seven structurally diverse isatin derivatives on P2X7-mediated IL-1β release from murine J774 macrophages. ATP-induced IL-1β and lactate dehydrogenase (LDH) release were assessed by specific colorimetric assays. P2X7 activity was determined by flow cytometric measurements of ATP-induced cation dye uptake. Cytotoxicity of isatin derivatives was determined using a tetrazolium-based colorimetric assay. ATP caused rapid IL-1β release in a concentration-dependent manner, and this process was completely impaired by the P2X7 antagonist, AZ10606120. In contrast, 5,7-dibromo-N-(p-methoxybenzyl)isatin (NAI) and 3-{4-[5,7-dibromo-1-(4-methoxybenzyl)-2-oxoindolin-3-ylidenamino]phenyl}propanoic acid (NAI-imine) enhanced P2X7-induced IL-1β release by twofold compared to that of isatin and the parent molecule, 5,7-dibromoisatin. NAI and NAI-imine had minimal effect on P2X7-induced dye uptake and LDH release. In contrast, 24-hour incubation with NAI and NAI-imine (in the absence of exogenous ATP) induced macrophage death in a concentration-dependent manner. In conclusion, this study demonstrates that N-alkyl-substituted isatins enhance P2X7 receptor-induced IL-1β release from murine macrophages. Thus, in addition to direct anticancer effects, these compounds may also impact inflammatory and immune cells within the tumor microenvironment. PMID:27524862

  6. Cathelicidin antimicrobial peptide inhibits fibroblast migration via P2X7 receptor signaling.

    PubMed

    Kumagai, Shohei; Matsui, Kazuki; Kawaguchi, Haruyo; Yamashita, Tomomi; Mohri, Tomomi; Fujio, Yasushi; Nakayama, Hiroyuki

    2013-08-01

    Fibrosis is one of the most common pathological alterations in heart failure, and fibroblast migration is an essential process in the development of cardiac fibrosis. Experimental autoimmune myocarditis (EAM) is a model of inflammatory heart disease characterized by inflammatory cell infiltration followed by healing without residual fibrosis. However, the precise mechanisms mediating termination of inflammation and nonfibrotic healing remain to be elucidated. Microarray analysis of hearts from model mice at multiple time points after EAM induction identified several secreted proteins upregulated during nonfibrotic healing, including the anti-inflammatory cathelicidin antimicrobial peptide (CAMP). Treatment with LL-37, a human homolog of CAMP, activated MAP kinases in fibroblasts but not in cardiomyocytes, indicating that fibroblasts were the target of CAMP activity. In addition, LL-37 decreased fibroblast migration in the in vitro scratch assay. P2X7 receptor (P2X7R), a well-known receptor for LL-37, was involved in LL-37 mediated biological effect on cardiac fibroblasts. Stimulation of BzATP, a P2X7R agonist, activated MAPK in fibroblasts, whereas the P2X7R antagonist, BBG, as well as P2X7R deletion abolished both LL-37-mediated MAPK activation and LL-37-induced reduction in fibroblast migration. These results strongly suggest that CAMP upregulation during myocarditis prevents myocardial fibrosis by restricting fibroblast migration via activation of the P2X7R-MAPK signaling pathway. PMID:23867818

  7. Structure-based identification and characterisation of structurally novel human P2X7 receptor antagonists.

    PubMed

    Caseley, Emily A; Muench, Stephen P; Fishwick, Colin W; Jiang, Lin-Hua

    2016-09-15

    The P2X7 receptor (P2X7R) plays an important role in diverse conditions associated with tissue damage and inflammation, meaning that the human P2X7R (hP2X7R) is an attractive therapeutic target. The crystal structures of the zebrafish P2X4R in the closed and ATP-bound open states provide an unprecedented opportunity for structure-guided identification of new ligands. The present study performed virtual screening of ∼100,000 structurally diverse compounds against the ATP-binding pocket in the hP2X7R. This identified three compounds (C23, C40 and C60) out of 73 top-ranked compounds by testing against hP2X7R-mediated Ca(2+) responses. These compounds were further characterised using Ca(2+) imaging, patch-clamp current recording, YO-PRO-1 uptake and propidium iodide cell death assays. All three compounds inhibited BzATP-induced Ca(2+) responses concentration-dependently with IC50s of 5.1±0.3μM, 4.8±0.8μM and 3.2±0.2μM, respectively. C23 and C40 inhibited BzATP-induced currents in a reversible and concentration-dependent manner, with IC50s of 0.35±0.3μM and 1.2±0.1μM, respectively, but surprisingly C60 did not affect BzATP-induced currents up to 100μM. They suppressed BzATP-induced YO-PRO-1 uptake with IC50s of 1.8±0.9μM, 1.0±0.1μM and 0.8±0.2μM, respectively. Furthermore, these three compounds strongly protected against ATP-induced cell death. Among them, C40 and C60 exhibited strong specificity towards the hP2X7R over the hP2X4R and rP2X3R. In conclusion, our study reports the identification of three novel hP2X7R antagonists with micromolar potency for the first time using a structure-based approach, including the first P2X7R antagonist with preferential inhibition of large pore formation. PMID:27481062

  8. Purinergic control of inflammation and thrombosis: Role of P2X1 receptors

    PubMed Central

    Oury, Cécile; Lecut, Christelle; Hego, Alexandre; Wéra, Odile; Delierneux, Céline

    2014-01-01

    Inflammation shifts the hemostatic mechanisms in favor of thrombosis. Upon tissue damage or infection, a sudden increase of extracellular ATP occurs, that might contribute to the crosstalk between inflammation and thrombosis. On platelets, P2X1 receptors act to amplify platelet activation and aggregation induced by other platelet agonists. These receptors critically contribute to thrombus stability in small arteries. Besides platelets, studies by our group indicate that these receptors are expressed by neutrophils. They promote neutrophil chemotaxis, both in vitro and in vivo. In a laser-induced injury mouse model of thrombosis, it appears that neutrophils are required to initiate thrombus formation and coagulation activation on inflamed arteriolar endothelia. In this model, by using P2X1−/ − mice, we recently showed that P2X1 receptors, expressed on platelets and neutrophils, play a key role in thrombus growth and fibrin generation. Intriguingly, in a model of endotoxemia, P2X1−/ − mice exhibited aggravated oxidative tissue damage, along with exacerbated thrombocytopenia and increased activation of coagulation, which translated into higher susceptibility to septic shock. Thus, besides its ability to recruit neutrophils and platelets on inflamed endothelia, the P2X1 receptor also contributes to limit the activation of circulating neutrophils under systemic inflammatory conditions. Taken together, these data suggest that P2X1 receptors are involved in the interplay between platelets, neutrophils and thrombosis. We propose that activation of these receptors by ATP on neutrophils and platelets represents a new mechanism that regulates thrombo-inflammation. PMID:25709760

  9. The P2X4 receptor is required for neuroprotection via ischemic preconditioning

    PubMed Central

    Ozaki, Tomohiko; Muramatsu, Rieko; Sasai, Miwa; Yamamoto, Masahiro; Kubota, Yoshiaki; Fujinaka, Toshiyuki; Yoshimine, Toshiki; Yamashita, Toshihide

    2016-01-01

    Ischemic preconditioning (IPC), a procedure consisting of transient ischemia and subsequent reperfusion, provides ischemic tolerance against prolonged ischemia in the brain. Although the blood flow changes mediated by IPC are primarily perceived by vascular endothelial cells, the role of these cells in ischemic tolerance has not been fully clarified. In this study, we found that the P2X4 receptor, which is abundantly expressed in vascular endothelial cells, is required for ischemic tolerance following middle artery occlusion (MCAO) in mice. Mechanistically, the P2X4 receptor was stimulated by fluid shear stress, which mimics reperfusion, thus promoting the increased expression of osteopontin, a neuroprotective molecule. Furthermore, we found that the intracerebroventricular administration of osteopontin was sufficient to exert a neuroprotective effect mediated by preconditioning-stimulated P2X4 receptor activation. These results demonstrate a novel mechanism whereby vascular endothelial cells are involved in ischemic tolerance. PMID:27173846

  10. Electroacupuncture at He-Mu points reduces P2X4 receptor expression in visceral hypersensitivity

    PubMed Central

    Guo, Xinxin; Chen, Jifei; Lu, Yuan; Wu, Luyi; Weng, Zhijun; Yang, Ling; Xin, Yuhu; Lin, Xianming; Liang, Yi; Fang, Jianqiao

    2013-01-01

    Electroacupuncture at Shangjuxu (ST37) and Tianshu (ST25) was reported to improve visceral hypersensitivity in rats. Colorectal distension was utilized to generate a rat model of chronic visceral hypersensitivity in irritable bowel syndrome. Results showed that abdominal withdrawal reflex scores noticeably increased after model establishment. Simultaneously, P2X4 receptor immureactivity significantly increased in the colon and spinal cord. Electroacupuncture and pinaverium bromide therapy both markedly decreased abdominal withdrawal reflex scores in rats with visceral hypersensitivity, and significantly decreased P2X4 receptor immunoreactivity in the colon and spinal cord. These data suggest that electroacupuncture treatment can improve visceral hypersensitivity in rats with irritable bowel syndrome by diminishing P2X4 receptor immunoreactivity in the colon and spinal cord. PMID:25206515

  11. Electroacupuncture at He-Mu points reduces P2X4 receptor expression in visceral hypersensitivity.

    PubMed

    Guo, Xinxin; Chen, Jifei; Lu, Yuan; Wu, Luyi; Weng, Zhijun; Yang, Ling; Xin, Yuhu; Lin, Xianming; Liang, Yi; Fang, Jianqiao

    2013-08-01

    Electroacupuncture at Shangjuxu (ST37) and Tianshu (ST25) was reported to improve visceral hypersensitivity in rats. Colorectal distension was utilized to generate a rat model of chronic visceral hypersensitivity in irritable bowel syndrome. Results showed that abdominal withdrawal reflex scores noticeably increased after model establishment. Simultaneously, P2X4 receptor immureactivity significantly increased in the colon and spinal cord. Electroacupuncture and pinaverium bromide therapy both markedly decreased abdominal withdrawal reflex scores in rats with visceral hypersensitivity, and significantly decreased P2X4 receptor immunoreactivity in the colon and spinal cord. These data suggest that electroacupuncture treatment can improve visceral hypersensitivity in rats with irritable bowel syndrome by diminishing P2X4 receptor immunoreactivity in the colon and spinal cord. PMID:25206515

  12. P2X7 Receptor Modulates Inflammatory and Functional Pulmonary Changes Induced by Silica

    PubMed Central

    Santana, Patrícia T.; Vieira, Flávia S.; da Graça, Carolyne Lalucha A. L.; Marques-da-Silva, Camila; Machado, Mariana N.; Caruso-Neves, Celso; Zin, Walter A.; Borojevic, Radovan; Coutinho-Silva, Robson

    2014-01-01

    Silicosis is an occupational lung disease, characterized by irreversible and progressive fibrosis. Silica exposure leads to intense lung inflammation, reactive oxygen production, and extracellular ATP (eATP) release by macrophages. The P2X7 purinergic receptor is thought to be an important immunomodulator that responds to eATP in sites of inflammation and tissue damage. The present study investigates the role of P2X7 receptor in a murine model of silicosis. To that end wild-type (C57BL/6) and P2X7 receptor knockout mice received intratracheal injection of saline or silica particles. After 14 days, changes in lung mechanics were determined by the end-inflation occlusion method. Bronchoalveolar lavage and flow cytometry analyzes were performed. Lungs were harvested for histological and immunochemistry analysis of fibers content, inflammatory infiltration, apoptosis, as well as cytokine and oxidative stress expression. Silica particle effects on lung alveolar macrophages and fibroblasts were also evaluated in cell line cultures. Phagocytosis assay was performed in peritoneal macrophages. Silica exposure increased lung mechanical parameters in wild-type but not in P2X7 knockout mice. Inflammatory cell infiltration and collagen deposition in lung parenchyma, apoptosis, TGF-β and NF-κB activation, as well as nitric oxide, reactive oxygen species (ROS) and IL-1β secretion were higher in wild-type than knockout silica-exposed mice. In vitro studies suggested that P2X7 receptor participates in silica particle phagocytosis, IL-1β secretion, as well as reactive oxygen species and nitric oxide production. In conclusion, our data showed a significant role for P2X7 receptor in silica-induced lung changes, modulating lung inflammatory, fibrotic, and functional changes. PMID:25310682

  13. Inhibition of antigen receptor-dependent Ca(2+) signals and NF-AT activation by P2X7 receptors in human B lymphocytes.

    PubMed

    Pippel, Anja; Beßler, Björn; Klapperstück, Manuela; Markwardt, Fritz

    2015-04-01

    One of the first intracellular signals after antigen binding by the antigen receptor of B lymphocytes is the increased intracellular Ca(2+) concentration ([Ca(2+)]i), which is followed by several intracellular signaling events like the nuclear translocation of the transcription factor NF-AT controlling the fate of B lymphocytes after their activation. Extracellular ATP, which is released from cells under several pathological conditions, is considered a danger-associated signal serving as an immunomodulator. We investigated the interaction of antigen receptor (BCR) and P2X7 receptor (P2X7R) activation on [Ca(2+)]i signaling and on nuclear translocation of the transcription factor NF-AT in human B lymphocytes. Although the P2X7R is an ATP-gated Ca(2+)-permeable ion channel, P2X7R activation inhibits the BCR-mediated [Ca(2+)]i responses. This effect is mimicked by cell membrane depolarization induced by an increase in the extracellular K(+) concentration or by application of the Na(+) ionophore gramicidin, but is abolished by stabilization of the membrane potential using the K(+) ionophore valinomycin, by extracellular Mg(2+), which is known to inhibit P2X7R-dependent effects, or by replacing Na(+) by the less P2X7R-permeable Tris(+) ion. Furthermore, P2X7R activation by ATP inhibits the BCR-dependent translocation of the transcription factor NF-ATc1 to the nucleus. We therefore conclude that extracellular ATP via the P2X7R mediates inhibitory effects on B cell activation. This may be of relevance for understanding of the activation of the BCR under pathological conditions and for the development of therapeutic strategies targeting human B lymphocytes or P2X7 receptors. PMID:25678443

  14. Blockade and reversal of spinal morphine tolerance by P2X3 receptor antagonist.

    PubMed

    Ma, Xiaqing; Xu, Tao; Xu, Hao; Jiang, Wei

    2015-04-01

    In recent years, studies have substantiated the view that P2X3 receptors play a part in the generation and transmission of purinergic signals in inflammatory and chronic neuropathic pain. Data have also been presented to suggest that the process of P2X3 receptor antagonism inhibits inflammatory hyperalgesia, involving the spinal opioid system. The aim of this study was to investigate the effect of the selective P2X3 receptor antagonist A-317491 on the development of antinociceptive tolerance to chronic morphine administration in mice. Daily systemic injection of A-317491 attenuated the morphine-induced antinociceptive tolerance to von Frey and thermal stimuli. Repeated morphine injections alone led to a significant rightward shift in the morphine dose-response curve compared with that with A-317491. A single dose of A-317491 also showed a reversal effect in morphine-tolerant mice. In a withdrawal test, co-administration of A-317491 and morphine also reduced the naloxone-induced withdrawal symptoms compared with the morphine-alone group. Thus, we propose that the P2X3 receptor is involved in the process of morphine antinociceptive tolerance and may be a new therapeutic target in the prevention of tolerance to morphine-induced antinociception. PMID:25350728

  15. Statins and ATP regulate nuclear pAkt via the P2X7 purinergic receptor in epithelial cells

    SciTech Connect

    Mistafa, Oras; Hoegberg, Johan; Stenius, Ulla

    2008-01-04

    Many studies have documented P2X7 receptor functions in cells of mesenchymal origin. P2X7 is also expressed in epithelial cells and its role in these cells remains largely unknown. Our data indicate that P2X7 regulate nuclear pAkt in epithelial cells. We show that low concentration of atorvastatin, a drug inhibiting HMG-CoA reductase and cholesterol metabolism, or the natural agonist extracellular ATP rapidly decreased the level of insulin-induced phosphorylated Akt in the nucleus. This effect was seen within minutes and was inhibited by P2X7 inhibitors. Experiments employing P2X7 siRNA and HEK293 cells heterologously expressing P2X7 and in vivo experiments further supported an involvement of P2X7. These data indicate that extracellular ATP and statins via the P2X7 receptor modulate insulin-induced Akt signaling in epithelial cells.

  16. Pirt reduces bladder overactivity by inhibiting purinergic receptor P2X3.

    PubMed

    Gao, Xiao-Fei; Feng, Ji-Feng; Wang, Wei; Xiang, Zheng-Hua; Liu, Xiu-Jie; Zhu, Chan; Tang, Zong-Xiang; Dong, Xin-Zhong; He, Cheng

    2015-01-01

    Pirt is a transmembrane protein predominantly expressed in peripheral neurons. However, the physiological and pathological roles of Pirt in hollow viscus are largely unknown. Here we show that Pirt deficiency in mice causes bladder overactivity. The density of α,β-meATP-induced currents is significantly reinforced in Pirt-deficient dorsal root ganglion (DRG) neurons. Pirt and P2X3 receptor co-localize in bladder nerve fibres and heterologous Pirt expression significantly reduces P2X3-mediated currents. Pirt interacts with P2X3 through the N-terminal 14 amino-acid residues. TAT-conjugated Pirt(N14) peptide (Pirt(N14)) is sufficient to inhibit P2X3 activation in bladder DRG neurons and to alleviate bladder overactivity in Pirt(-/-) mice. Pirt expression is decreased in the bladder of cyclophosphamide (CYP)-treated mice, a commonly used model of bladder overactivity. Importantly, Pirt(N14) administration reduces the frequency of bladder voiding and restores the voided volume of CYP-treated mice. Therefore, our results demonstrate that Pirt is an endogenous regulator of P2X3 in bladder function. PMID:26151598

  17. Photo-switchable tweezers illuminate pore-opening motions of an ATP-gated P2X ion channel

    PubMed Central

    Habermacher, Chloé; Martz, Adeline; Calimet, Nicolas; Lemoine, Damien; Peverini, Laurie; Specht, Alexandre; Cecchini, Marco; Grutter, Thomas

    2016-01-01

    P2X receptors function by opening a transmembrane pore in response to extracellular ATP. Recent crystal structures solved in apo and ATP-bound states revealed molecular motions of the extracellular domain following agonist binding. However, the mechanism of pore opening still remains controversial. Here we use photo-switchable cross-linkers as ‘molecular tweezers’ to monitor a series of inter-residue distances in the transmembrane domain of the P2X2 receptor during activation. These experimentally based structural constraints combined with computational studies provide high-resolution models of the channel in the open and closed states. We show that the extent of the outer pore expansion is significantly reduced compared to the ATP-bound structure. Our data further reveal that the inner and outer ends of adjacent pore-lining helices come closer during opening, likely through a hinge-bending motion. These results provide new insight into the gating mechanism of P2X receptors and establish a versatile strategy applicable to other membrane proteins. DOI: http://dx.doi.org/10.7554/eLife.11050.001 PMID:26808983

  18. Photo-switchable tweezers illuminate pore-opening motions of an ATP-gated P2X ion channel.

    PubMed

    Habermacher, Chloé; Martz, Adeline; Calimet, Nicolas; Lemoine, Damien; Peverini, Laurie; Specht, Alexandre; Cecchini, Marco; Grutter, Thomas

    2016-01-01

    P2X receptors function by opening a transmembrane pore in response to extracellular ATP. Recent crystal structures solved in apo and ATP-bound states revealed molecular motions of the extracellular domain following agonist binding. However, the mechanism of pore opening still remains controversial. Here we use photo-switchable cross-linkers as 'molecular tweezers' to monitor a series of inter-residue distances in the transmembrane domain of the P2X2 receptor during activation. These experimentally based structural constraints combined with computational studies provide high-resolution models of the channel in the open and closed states. We show that the extent of the outer pore expansion is significantly reduced compared to the ATP-bound structure. Our data further reveal that the inner and outer ends of adjacent pore-lining helices come closer during opening, likely through a hinge-bending motion. These results provide new insight into the gating mechanism of P2X receptors and establish a versatile strategy applicable to other membrane proteins. PMID:26808983

  19. Ionotropic purinergic receptors P2X in frog and turtle retina: glial and neuronal localization.

    PubMed

    Vitanova, Lily Alexandrova; Kupenova, Petia Nikolova

    2014-06-01

    Purinergic signaling is represented in both the peripheral and central nervous system (CNS), and in particular in the retina, which may be regarded as a part of the CNS. While purigenic signaling is relatively well studied in mammalian retinas, little is known about it in retinas of lower vertebrates. The aim of present study was to investigate, using immunocytochemistry, the distribution of purinoreceptors P2X in retinas of frog and turtle, which are appropriate models of the brain neuron-to-glia interactions. The results showed widespread expression of all seven ionotropic purinoreceptors (P2X1-P2X7) in both frog and turtle retinas. They were predominantly expressed in Müller cells, the principal glial cells in the retina. All structures typical of Müller cells: the outer and the inner limiting membranes, the cells bodies in the inner nuclear layer, the radial processes in the inner plexiform layer (IPL), and the so called endfeet (frog) or the orthogonal arrays of particles (turtle) in the ganglion cells layer were immunostained. Colocalizations between P2X1-P2X7 and the glial cell marker Vimentin proved that the immunostaining was in the Müller cells. In addition to the glial staining, neuronal staining was also seen as fine puncta in the inner plexiform layer and by small dots and patches in the outer plexiform layer. Some cell bodies of horizontal, amacrine and ganglion cells were also stained. The results obtained imply that the purinergic P2X receptors may significantly contribute to the neuron-to-glia signaling in retinas of the lower vertebrates. PMID:24461518

  20. ATP excites mouse vomeronasal sensory neurons through activation of P2X receptors.

    PubMed

    Vick, J S; Delay, R J

    2012-09-18

    Purinergic signaling through activation of P2X and P2Y receptors is critically important in the chemical senses. In the mouse main olfactory epithelium (MOE), adenosine 5'-triphosphate (ATP) elicits an increase in intracellular calcium ([Ca(2+)](I)) and reduces the responsiveness of olfactory sensory neurons to odorants through activation of P2X and P2Y receptors. We investigated the role of purinergic signaling in vomeronasal sensory neuron (VSN)s from the mouse vomeronasal organ (VNO), an olfactory organ distinct from the MOE that responds to many conspecific chemical cues. Using a combination of calcium imaging and patch-clamp electrophysiology with isolated VSNs, we demonstrated that ATP elicits an increase in [Ca(2+)](I) and an inward current with similar EC(50)s. Neither adenosine nor the P2Y receptor ligands adenosine 5'-diphosphate, uridine 5'-triphosphate, and uridine-5'-disphosphate could mimic either effect of ATP. Moreover, the increase in [Ca(2+)](I) required the presence of extracellular calcium and the inward current elicited by ATP was partially blocked by the P2X receptor antagonists pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate and 2',3'-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate. Consistent with the activation of P2X receptors, we detected gene expression of the P2X1 and 3 receptors in the VNO by Reverse transcription polymerase chain reaction (RT-PCR). When co-delivered with dilute urine, a natural stimulus, ATP significantly increased the inward current above that elicited by dilute urine or ATP alone. Mechanical stimulation of the VNO induced the release of ATP, detected by luciferin-luciferase luminometry, and this release of ATP was completely abolished in the presence of the connexin/pannexin hemichannel blocker, carbenoxolone. We conclude that the release of ATP could occur during the activity of the vasomotor pump that facilitates the movement of chemicals into the VNO for detection by VSNs. This mechanism could lead to a

  1. A Dual Role for P2X7 Receptor during Porphyromonas gingivalis Infection.

    PubMed

    Ramos-Junior, E S; Morandini, A C; Almeida-da-Silva, C L C; Franco, E J; Potempa, J; Nguyen, K A; Oliveira, A C; Zamboni, D S; Ojcius, D M; Scharfstein, J; Coutinho-Silva, R

    2015-09-01

    Emerging evidence suggests a role for purinergic signaling in the activation of multiprotein intracellular complexes called inflammasomes, which control the release of potent inflammatory cytokines, such as interleukin (IL) -1β and -18. Porphyromonas gingivalis is intimately associated with periodontitis and is currently considered one of the pathogens that can subvert the immune system by limiting the activation of the NLRP3 inflammasome. We recently showed that P. gingivalis can dampen eATP-induced IL-1β secretion by means of its fimbriae in a purinergic P2X7 receptor-dependent manner. Here, we further explore the role of this purinergic receptor during eATP-induced IL-1β processing and secretion by P. gingivalis-infected macrophages. We found that NLRP3 was necessary for eATP-induced IL-1β secretion as well as for caspase 1 activation irrespective of P. gingivalis fimbriae. Additionally, although the secretion of IL-1β from P. gingivalis-infected macrophages was dependent on NLRP3, its adaptor protein ASC, or caspase 1, the cleavage of intracellular pro-IL-1β to the mature form was found to occur independently of NLRP3, its adaptor protein ASC, or caspase 1. Our in vitro findings revealed that P2X7 receptor has a dual role, being critical not only for eATP-induced IL-1β secretion but also for intracellular pro-IL-1β processing. These results were relevant in vivo since P2X7 receptor expression was upregulated in a P. gingivalis oral infection model, and reduced IFN-γ and IL-17 were detected in draining lymph node cells from P2rx7(-/-) mice. Furthermore, we demonstrated that P2X7 receptor and NLRP3 transcription were modulated in human chronic periodontitis. Overall, we conclude that the P2X7 receptor has a role in periodontal immunopathogenesis and suggest that targeting of the P2X7/NLRP3 pathway should be considered in future therapeutic interventions in periodontitis. PMID:26152185

  2. Multiple Roles of the Extracellular Vestibule Amino Acid Residues in the Function of the Rat P2X4 Receptor

    PubMed Central

    Rokic, Milos B.; Stojilkovic, Stanko S.; Vavra, Vojtech; Kuzyk, Pavlo; Tvrdonova, Vendula; Zemkova, Hana

    2013-01-01

    The binding of ATP to trimeric P2X receptors (P2XR) causes an enlargement of the receptor extracellular vestibule, leading to opening of the cation-selective transmembrane pore, but specific roles of vestibule amino acid residues in receptor activation have not been evaluated systematically. In this study, alanine or cysteine scanning mutagenesis of V47–V61 and F324–N338 sequences of rat P2X4R revealed that V49, Y54, Q55, F324, and G325 mutants were poorly responsive to ATP and trafficking was only affected by the V49 mutation. The Y54F and Y54W mutations, but not the Y54L mutation, rescued receptor function, suggesting that an aromatic residue is important at this position. Furthermore, the Y54A and Y54C receptor function was partially rescued by ivermectin, a positive allosteric modulator of P2X4R, suggesting a rightward shift in the potency of ATP to activate P2X4R. The Q55T, Q55N, Q55E, and Q55K mutations resulted in non-responsive receptors and only the Q55E mutant was ivermectin-sensitive. The F324L, F324Y, and F324W mutations also rescued receptor function partially or completely, ivermectin action on channel gating was preserved in all mutants, and changes in ATP responsiveness correlated with the hydrophobicity and side chain volume of the substituent. The G325P mutant had a normal response to ATP, suggesting that G325 is a flexible hinge. A topological analysis revealed that the G325 and F324 residues disrupt a β-sheet upon ATP binding. These results indicate multiple roles of the extracellular vestibule amino acid residues in the P2X4R function: the V49 residue is important for receptor trafficking to plasma membrane, the Y54 and Q55 residues play a critical role in channel gating and the F324 and G325 residues are critical for vestibule widening. PMID:23555667

  3. Lack of a Functioning P2X7 Receptor Leads to Increased Susceptibility to Toxoplasmic Ileitis

    PubMed Central

    Robinson, Dionne P.; Fuller, Stephen J.; Walker, Robert A.; Ikin, Rowan J.; Bao, Shisan J.; Grigg, Michael E.; Wiley, James S.; Smith, Nicholas C.

    2015-01-01

    Background Oral infection of C57BL/6J mice with the protozoan parasite Toxoplasma gondii leads to a lethal inflammatory ileitis. Principal Findings Mice lacking the purinergic receptor P2X7R are acutely susceptible to toxoplasmic ileitis, losing significantly more weight than C57BL/6J mice and exhibiting much greater intestinal inflammatory pathology in response to infection with only 10 cysts of T. gondii. This susceptibility is not dependent on the ability of P2X7R-deficient mice to control the parasite, which they accomplish just as efficiently as C57BL/6J mice. Rather, susceptibility is associated with elevated ileal concentrations of pro-inflammatory cytokines, reactive nitrogen intermediates and altered regulation of elements of NFκB activation in P2X7R-deficient mice. Conclusions Our data support the thesis that P2X7R, a well-documented activator of pro-inflammatory cytokine production, also plays an important role in the regulation of intestinal inflammation. PMID:26053862

  4. Blocking the P2X7 Receptor Improves Outcomes After Axonal Fusion

    PubMed Central

    Rodriguez-Feo, Charles L.; Sexton, Kevin W.; Boyer, Richard B.; Pollins, Alonda C.; Cardwell, Nancy L.; Nanney, Lillian B.; Shack, R. Bruce; Mikesh, Michelle A; McGill, Christopher H; Driscoll, Christopher W; Bittner, George D.; Thayer, Wesley P.

    2013-01-01

    Background Activation of the P2X7 Receptor on nerve cells causes the formation of pannexin pores, which allows the influx of calcium across the cell membrane. Polyethylene glycol (PEG) and methylene blue (MB) have previously been shown to delay Wallerian degeneration if applied during microsuture repair of the severed nerve. Our hypothesis is that by modulating calcium influx via the P2X7 receptor pathway, we could improve PEG based axonal repair. The P2X7 receptor can be stimulated or inhibited using bzATP or Brilliant Blue (FCF), respectively. Methods A single incision rat sciatic nerve injury model was used. The defect was repaired using a previously described PEG, MB fusion protocol. Experimental animals were treated with 100 µL of 100 µM FCF solution (n=8) or 100 µL of a 30 µM bzATP solution (n=6). Control animals received neither FCF, bzATP, nor PEG. Compound Action Potentials (CAPs) were recorded prior to transection (baseline), immediately after repair, and 21 days post operatively. Animals underwent behavioral testing 3,7, 14, and 21 days post operatively. After sacrifice, nerves were fixed, sectioned, and immunostained to allow for counting of total axons. Results Rats treated with FCF showed an improvement as compared to control at all time points (n=8) (p= .047, .044, .014, and .0059 respectively). A statistical difference was also shown between FCF and bzATP at Day 7 (p<.05), but not shown with days 3, 14, and 21. (p>.05). Conclusions Blocking the P2X7 receptor improves functional outcomes after PEG mediated axonal fusion. PMID:23731685

  5. Conductance of P2X4 purinergic receptor is determined by conformational equilibrium in the transmembrane region.

    PubMed

    Minato, Yuichi; Suzuki, Shiho; Hara, Tomoaki; Kofuku, Yutaka; Kasuya, Go; Fujiwara, Yuichiro; Igarashi, Shunsuke; Suzuki, Ei-Ichiro; Nureki, Osamu; Hattori, Motoyuki; Ueda, Takumi; Shimada, Ichio

    2016-04-26

    Ligand-gated ion channels are partially activated by their ligands, resulting in currents lower than the currents evoked by the physiological full agonists. In the case of P2X purinergic receptors, a cation-selective pore in the transmembrane region expands upon ATP binding to the extracellular ATP-binding site, and the currents evoked by α,β-methylene ATP are lower than the currents evoked by ATP. However, the mechanism underlying the partial activation of the P2X receptors is unknown although the crystal structures of zebrafish P2X4 receptor in the apo and ATP-bound states are available. Here, we observed the NMR signals from M339 and M351, which were introduced in the transmembrane region, and the endogenous alanine and methionine residues of the zebrafish P2X4 purinergic receptor in the apo, ATP-bound, and α,β-methylene ATP-bound states. Our NMR analyses revealed that, in the α,β-methylene ATP-bound state, M339, M351, and the residues that connect the ATP-binding site and the transmembrane region, M325 and A330, exist in conformational equilibrium between closed and open conformations, with slower exchange rates than the chemical shift difference (<100 s(-1)), suggesting that the small population of the open conformation causes the partial activation in this state. Our NMR analyses also revealed that the transmembrane region adopts the open conformation in the state bound to the inhibitor trinitrophenyl-ATP, and thus the antagonism is due to the closure of ion pathways, except for the pore in the transmembrane region: i.e., the lateral cation access in the extracellular region. PMID:27071117

  6. P2X and P2Y receptor signaling in red blood cells

    PubMed Central

    Sluyter, Ronald

    2015-01-01

    Purinergic signaling involves the activation of cell surface P1 and P2 receptors by extracellular nucleosides and nucleotides such as adenosine and adenosine triphosphate (ATP), respectively. P2 receptors comprise P2X and P2Y receptors, and have well-established roles in leukocyte and platelet biology. Emerging evidence indicates important roles for these receptors in red blood cells. P2 receptor activation stimulates a number of signaling pathways in progenitor red blood cells resulting in microparticle release, reactive oxygen species formation, and apoptosis. Likewise, activation of P2 receptors in mature red blood cells stimulates signaling pathways mediating volume regulation, eicosanoid release, phosphatidylserine exposure, hemolysis, impaired ATP release, and susceptibility or resistance to infection. This review summarizes the distribution of P2 receptors in red blood cells, and outlines the functions of P2 receptor signaling in these cells and its implications in red blood cell biology. PMID:26579528

  7. Predictions Suggesting a Participation of β-Sheet Configuration in the M2 Domain of the P2X7 Receptor: A Novel Conformation?

    PubMed Central

    Teixeira, Pedro Celso Nogueira; de Souza, Cristina Alves Magalhães; de Freitas, Mônica Santos; Foguel, Débora; Caffarena, Ernesto Raul; Alves, Luiz Anastacio

    2009-01-01

    Scanning experiments have shown that the putative TM2 domain of the P2X7 receptor (P2X7R) lines the ionic pore. However, none has identified an α-helix structure, the paradigmatic secondary structure of ion channels in mammalian cells. In addition, some researchers have suggested a β-sheet conformation in the TM2 domain of P2X2. These data led us to investigate a new architecture within the P2X receptor family. P2X7R is considered an intriguing receptor because its activation induces nonselective large pore formation, in contrast to the majority of other ionic channel proteins in mammals. This receptor has two states: a low-conductance channel (∼10 pS) and a large pore (>400 pS). To our knowledge, one fundamental question remains unanswered: Are the P2X7R channel and the pore itself the same entity or are they different structures? There are no structural data to help solve this question. Thus, we investigated the hydrophobic M2 domain with the aim of predicting the fitted position and the secondary structure of the TM2 segment from human P2X7R (hP2X7R). We provide evidence for a β-sheet conformation, using bioinformatics algorithms and molecular-dynamics simulation in conjunction with circular dichroism in different environments and Fourier transform infrared spectroscopy. In summary, our study suggests the possibility that a segment composed of residues from part of the M2 domain and part of the putative TM2 segment of P2X7R is partially folded in a β-sheet conformation, and may play an important role in channel/pore formation associated with P2X7R activation. It is important to note that most nonselective large pores have a transmembrane β-sheet conformation. Thus, this study may lead to a paradigmatic change in the P2X7R field and/or raise new questions about this issue. PMID:19186133

  8. Electroacupuncture and A-317491 depress the transmission of pain on primary afferent mediated by the P2X3 receptor in rats with chronic neuropathic pain states.

    PubMed

    Wang, Wan-Sheng; Tu, Wen-Zhan; Cheng, Rui-Dong; He, Rong; Ruan, Li-Hua; Zhang, Li; Gong, Yong-Sheng; Fan, Xiao-Fang; Hu, Jie; Cheng, Bo; Lai, Yin-Ping; Zou, En-Miao; Jiang, Song-He

    2014-12-01

    P2X is a family of ligand-gated ion channels that act through adenosine ATP. The P2X3 receptor plays a key role in the transmission of neuropathic pain at peripheral and spinal sites. Electroacupuncture (EA) has been used to treat neuropathic pain effectively. To determine the role of EA in neuropathic pain mediated through the P2X3 receptor in dorsal root ganglion neurons and the spinal cord, a chronic constriction injury (CCI) model was used. Sprague-Dawley rats were divided into four groups: sham CCI, CCI, CCI plus contralateral EA, and CCI plus ipsilateral EA. The mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were recorded. Furthermore, the expression of the P2X3 receptor was evaluated through Western blotting and immunofluorescence. The effects of EA and A-317491 were investigated through the whole-cell patch-clamp method and intrathecal administration. Our results show that the MWT and TWL of EA groups were higher than those in the CCI group, whereas the expression of the P2X3 receptor was lower than that in the CCI group. However, no significant difference was detected between the two EA groups. EA depressed the currents created by ATP and the upregulation of the P2X3 receptor in CCI rats. Additionally, EA was more potent in reducing mechanical allodynia and thermal hyperalgesia when combined with A-317491 through intrathecal administration. These results show that both contralateral and ipsilateral EA might inhibit the primary afferent transmission of neuropathic pain induced through the P2X3 receptor. In addition, EA and A-317491 might have an additive effect in inhibiting the transmission of pain mediated by the P2X3 receptor. PMID:25041872

  9. Functional polymorphisms in the P2X7 receptor gene are associated with stress fracture injury.

    PubMed

    Varley, Ian; Greeves, Julie P; Sale, Craig; Friedman, Eitan; Moran, Daniel S; Yanovich, Ran; Wilson, Peter J; Gartland, Alison; Hughes, David C; Stellingwerff, Trent; Ranson, Craig; Fraser, William D; Gallagher, James A

    2016-03-01

    Military recruits and elite athletes are susceptible to stress fracture injuries. Genetic predisposition has been postulated to have a role in their development. The P2X7 receptor (P2X7R) gene, a key regulator of bone remodelling, is a genetic candidate that may contribute to stress fracture predisposition. The aim of this study is to evaluate the putative contribution of P2X7R to stress fracture injury in two separate cohorts, military personnel and elite athletes. In 210 Israeli Defense Forces (IDF) military conscripts, stress fracture injury was diagnosed (n = 43) based on symptoms and a positive bone scan. In a separate cohort of 518 elite athletes, self-reported medical imaging scan-certified stress fracture injuries were recorded (n = 125). Non-stress fracture controls were identified from these cohorts who had a normal bone scan or no history or symptoms of stress fracture injury. Study participants were genotyped for functional SNPs within the P2X7R gene using proprietary fluorescence-based competitive allele-specific PCR assay. Pearson's chi-squared (χ (2)) tests, corrected for multiple comparisons, were used to assess associations in genotype frequencies. The variant allele of P2X7R SNP rs3751143 (Glu496Ala-loss of function) was associated with stress fracture injury, whilst the variant allele of rs1718119 (Ala348Thr-gain of function) was associated with a reduced occurrence of stress fracture injury in military conscripts (P < 0.05). The association of the variant allele of rs3751143 with stress fractures was replicated in elite athletes (P < 0.05), whereas the variant allele of rs1718119 was also associated with reduced multiple stress fracture cases in elite athletes (P < 0.05). The association between independent P2X7R polymorphisms with stress fracture prevalence supports the role of a genetic predisposition in the development of stress fracture injury. PMID:26825304

  10. [ROLE PHOSPHOINOSITID SIGNALING PATHWAY IN OPIOIDS CONTROL OF P2X3 RECEPTORS IN THE PRIMARY SENSORY NEURONS].

    PubMed

    Kulyk, V B; Chizhmakov, I V; Volkova, T M; Maximyuk, O P; Krishtal, O A

    2015-01-01

    Homomeric P2X3 receptors expressed in primary nociceptive neurons are crucial elements in the pain signal generation. In turn, opioid system regulates the intensity of this signal in both CNS and PNS. Here we describe the effects of opioids on P2X3 receptors in DRG neurons studied by using patch clamp technique. Activation of G-protein coupled opioid receptors by endogenous opioid Leu-enkephalin (Leu), resulted in the two opposite effects on P2X3 receptor-mediated currents (P2X3 currents). In particular, application of 1 µM Leu lead to the complete inhibition of P2X3 currents. However, after pretreatment of the neurons with a Gi/o-protein inhibitor pertussis toxin (PT), the same concentration of Leu caused facilitation of P2X3 currents. PLC inhibitor U-73122 at concentration of 1 µM completely eliminated both facilitating and inhibitory effects of Leu on P2X3 currents. Thus, opioid receptor agonists cause two oppositely directed effects on P2X3 receptors in DRG neurons of rats and both of them are mediated through PLC signaling pathway. Our results point to a possible molecular basis of the mechanism for the well-known transition inhibitory action of opioids (analgesia) to facilitating (hyperalgesia). PMID:26552301

  11. Peripheral and central P2X3 receptor contributions to colon mechanosensitivity and hypersensitivity in the mouse

    PubMed Central

    Shinoda, Masamichi; Feng, Bin; Gebhart, G. F.

    2009-01-01

    Background & Aims Irritable bowel syndrome is characterized by altered sensory qualities, namely discomfort/pain and colorectal hypersensitivity. In mice, we examined the role of P2X3 receptors in colon mechanosensitivity and intracolonic zymosan-produced hypersensitivity, a model of persistent colon hypersensitivity without colon inflammation. Methods The visceromotor response (VMR) to colon distension (15 – 60 mmHg) was determined before and after intracolonic saline or zymosan (30 mg/mL, 0.1 mL, daily for 3 days) treatment. Colon pathology and intracolonic ATP release was assessed in parallel experiments. To examine P2X3 receptor contributions to colon mechanosensation and hypersensitivity, electrophysiological experiments were performed using an in vitro colon-pelvic nerve preparation. Results VMRs to distension were significantly reduced in P2X3+/−and P2X3−/− mice relative to wildtype mice. Colon hypersensitivity produced by zymosan was virtually absent in P2X3−/− relative to wildtype or P2X3+/− mice. Intralumenal release of the endogenous P2X receptor ligand ATP did not differ between wildtype and P2X3−/− mice or change after intracolonic zymosan treatment. Responses of muscular and muscular-mucosal pelvic nerve afferents to mechanical stretch did not differ between P2X3−/− and wildtype mice. Both muscular and muscular-mucosal afferents in wildtype mice sensitized to application of an inflammatory soup, whereas only muscular-mucosal afferents did so in P2X3−/− mice. Conclusions These results suggest differential roles for peripheral and central P2X3 receptors in colon mechanosensory transduction and hypersensitivity. PMID:19549524

  12. Promoted Interaction of Nuclear Factor-κB With Demethylated Purinergic P2X3 Receptor Gene Contributes to Neuropathic Pain in Rats With Diabetes.

    PubMed

    Zhang, Hong-Hong; Hu, Ji; Zhou, You-Lang; Qin, Xin; Song, Zhen-Yuan; Yang, Pan-Pan; Hu, Shufen; Jiang, Xinghong; Xu, Guang-Yin

    2015-12-01

    Painful diabetic neuropathy is a common complication of diabetes produced by mechanisms that as yet are incompletely defined. The aim of this study was to investigate the roles of nuclear factor-κB (NF-κB) in the regulation of purinergic receptor P2X ligand-gated ion channel 3 (P2X3R) plasticity in dorsal root ganglion (DRG) neurons of rats with painful diabetes. Here, we showed that hindpaw pain hypersensitivity in streptozocin-induced diabetic rats was attenuated by treatment with purinergic receptor antagonist suramin or A-317491. The expression and function of P2X3Rs was markedly enhanced in hindpaw-innervated DRG neurons in diabetic rats. The CpG (cytosine guanine dinucleotide) island in the p2x3r gene promoter region was significantly demethylated, and the expression of DNA methyltransferase 3b was remarkably downregulated in DRGs in diabetic rats. The binding ability of p65 (an active form of NF-κB) with the p2x3r gene promoter region and p65 expression were enhanced significantly in diabetes. The inhibition of p65 signaling using the NF-κB inhibitor pyrrolidine dithiocarbamate or recombinant lentiviral vectors designated as lentiviral vector-p65 small interfering RNA remarkably suppressed P2X3R activities and attenuated diabetic pain hypersensitivity. Insulin treatment significantly attenuated pain hypersensitivity and suppressed the expression of p65 and P2X3Rs. Our findings suggest that the p2x3r gene promoter DNA demethylation and enhanced interaction with p65 contributes to P2X3R sensitization and diabetic pain hypersensitivity. PMID:26130762

  13. Functional expression of P2X family receptors in macrophages is affected by microenvironment in mouse T cell acute lymphoblastic leukemia

    SciTech Connect

    Chen, Shayan; Feng, Wenli; Yang, Xiao; Yang, Wanzhu; Ru, Yongxin; Liao, Jinfeng; Wang, Lina; Lin, Yongmin; Ren, Qian; Zheng, Guoguang

    2014-04-18

    Highlights: • We study the impact of leukemic microenvironment on P2X family receptors in Mφs. • Bone marrow and spleen Mφs are studied in Notch1-induced mouse leukemia model. • Increased expression of P2X7R is found in Mφs during the development of leukemia. • Elevated P2X7R-mediated calcium response is found in Mφs at late stage of leukemia. • More apoptotic Mφs are found in bone marrow and spleen at late stage of leukemia. - Abstract: Nucleotides are important players in intercellular signaling communication network. P2X family receptors (P2XRs) are ATP-gated plasma membrane ion channels with diverse biological functions. Macrophages are important components in the microenvironment of hematopoiesis participating in both physiological and pathological processes. However, the role of P2XRs in macrophages in leukemia has not been established. Here we investigated expression pattern and functions of P2XRs in macrophages from bone marrow (BM) and spleen of Notch1-induced T-ALL mice. Real-time PCR showed that P2XRs except P2X5R were expressed in BM and spleen macrophages. Furthermore, with the development of leukemia, the expression of P2X7R increased in both BM and spleen macrophages whereas expression of P2X1R increased in spleen macrophages. Live cell imaging recoding the Ca{sup 2+} response demonstrated that P2X7R expressed in macrophages was functional. TUNEL and electron microscopy analysis found that apoptotic macrophages were frequently observed in BM and spleen at late stage of leukemia, which was partly contributed by the activation of overexpressed P2X7R. Our results suggested that the intercellular communication mediated by nucleotides might orchestrate in the pathological process of leukemia and could be a potential target for the treatment of leukemia.

  14. Modulation of Mouse Embryonic Stem Cell Proliferation and Neural Differentiation by the P2X7 Receptor

    PubMed Central

    Glaser, Talita; de Oliveira, Sophia La Banca; Cheffer, Arquimedes; Beco, Renata; Martins, Patrícia; Fornazari, Maynara; Lameu, Claudiana; Junior, Helio Miranda Costa; Coutinho-Silva, Robson; Ulrich, Henning

    2014-01-01

    Background Novel developmental functions have been attributed to the P2X7 receptor (P2X7R) including proliferation stimulation and neural differentiation. Mouse embryonic stem cells (ESC), induced with retinoic acid to neural differentiation, closely assemble processes occurring during neuroectodermal development of the early embryo. Principal Findings P2X7R expression together with the pluripotency marker Oct-4 was highest in undifferentiated ESC. In undifferentiated cells, the P2X7R agonist Bz-ATP accelerated cell cycle entry, which was blocked by the specific P2X7R inhibitor KN-62. ESC induced to neural differentiation with retinoic acid, reduced Oct-4 and P2X7R expression. P2X7R receptor-promoted intracellular calcium fluxes were obtained at lower Bz-ATP ligand concentrations in undifferentiated and in neural-differentiated cells compared to other studies. The presence of KN-62 led to increased number of cells expressing SSEA-1, Dcx and β3-tubulin, as well as the number of SSEA-1 and β3-tubulin-double-positive cells confirming that onset of neuroectodermal differentiation and neuronal fate determination depends on suppression of P2X7R activity. Moreover, an increase in the number of Ki-67 positive cells in conditions of P2X7R inhibition indicates rescue of progenitors into the cell cycle, augmenting the number of neuroblasts and consequently neurogenesis. Conclusions In embryonic cells, P2X7R expression and activity is upregulated, maintaining proliferation, while upon induction to neural differentiation P2X7 receptor expression and activity needs to be suppressed. PMID:24798220

  15. The microglial ATP-gated ion channel P2X7 as a CNS drug target.

    PubMed

    Bhattacharya, Anindya; Biber, Knut

    2016-10-01

    Based on promising preclinical evidence, microglial P2X7 has increasingly being recognized as a target for therapeutic intervention in neurological and psychiatric diseases. However, despite this knowledge no P2X7-related drug has yet entered clinical trials with respect to CNS diseases. We here discuss the current literature on P2X7 being a drug target and identify unsolved issues and still open questions that have hampered the development of P2X7 dependent therapeutic approaches for CNS diseases. It is concluded here that the lack of brain penetrating P2X7 antagonists is a major obstacle in the field and that central P2X7 is a yet untested clinical drug target. In the CNS, microglial P2X7 activation causes neuroinflammation, which in turn plays a role in various CNS disorders. This has resulted in a surge of brain penetrant P2X7 antagonists. P2X7 is a viable, clinically untested CNS drug target. GLIA 2016;64:1772-1787. PMID:27219534

  16. Stable, synthetic analogs of diadenosine tetraphosphate inhibit rat and human P2X3 receptors and inflammatory pain

    PubMed Central

    Viatchenko-Karpinski, Viacheslav; Novosolova, Natalia; Ishchenko, Yevheniia; Azhar, M Ameruddin; Wright, Michael; Tsintsadze, Vera; Kamal, Ahmed; Burnashev, Nail; Voitenko, Nana; Giniatullin, Rashid; Lozovaya, Natalia

    2016-01-01

    Background A growing body of evidence suggests that ATP-gated P2X3 receptors (P2X3Rs) are implicated in chronic pain. We address the possibility that stable, synthetic analogs of diadenosine tetraphosphate (Ap4A) might induce antinociceptive effects by inhibiting P2X3Rs in peripheral sensory neurons. Results The effects of two stable, synthetic Ap4A analogs (AppNHppA and AppCH2ppA) are studied firstly in vitro on HEK293 cells expressing recombinant rat P2XRs (P2X2Rs, P2X3Rs, P2X4Rs, and P2X7Rs) and then using native rat brain cells (cultured trigeminal, nodose, or dorsal root ganglion neurons). Thereafter, the action of these stable, synthetic Ap4A analogs on inflammatory pain and thermal hyperalgesia is studied through the measurement of antinociceptive effects in formalin and Hargreaves plantar tests in rats in vivo. In vitro inhibition of rat P2X3Rs (not P2X2Rs, P2X4Rs nor P2X7Rs) is shown to take place mediated by high-affinity desensitization (at low concentrations; IC50 values 100–250 nM) giving way to only weak partial agonism at much higher concentrations (EC50 values ≥ 10 µM). Similar inhibitory activity is observed with human recombinant P2X3Rs. The inhibitory effects of AppNHppA on nodose, dorsal root, and trigeminal neuron whole cell currents suggest that stable, synthetic Ap4A analogs inhibit homomeric P2X3Rs in preference to heteromeric P2X2/3Rs. Both Ap4A analogs mediate clear inhibition of pain responses in both in vivo inflammation models. Conclusions Stable, synthetic Ap4A analogs (AppNHppA and AppCH2ppA) being weak partial agonist provoke potent high-affinity desensitization-mediated inhibition of homomeric P2X3Rs at low concentrations. Therefore, both analogs demonstrate clear potential as potent analgesic agents for use in the management of chronic pain associated with heightened P2X3R activation. PMID:27030723

  17. Neutrophil P2X7 receptors mediate NLRP3 inflammasome-dependent IL-1β secretion in response to ATP

    PubMed Central

    Karmakar, Mausita; Katsnelson, Michael A.; Dubyak, George R.; Pearlman, Eric

    2016-01-01

    Although extracellular ATP is abundant at sites of inflammation, its role in activating inflammasome signalling in neutrophils is not well characterized. In the current study, we demonstrate that human and murine neutrophils express functional cell-surface P2X7R, which leads to ATP-induced loss of intracellular K+, NLRP3 inflammasome activation and IL-1β secretion. ATP-induced P2X7R activation caused a sustained increase in intracellular [Ca2+], which is indicative of P2X7R channel opening. Although there are multiple polymorphic variants of P2X7R, we found that neutrophils from multiple donors express P2X7R, but with differential efficacies in ATP-induced increase in cytosolic [Ca2+]. Neutrophils were also the predominant P2X7R-expressing cells during Streptococcus pneumoniae corneal infection, and P2X7R was required for bacterial clearance. Given the ubiquitous presence of neutrophils and extracellular ATP in multiple inflammatory conditions, ATP-induced P2X7R activation and IL-1β secretion by neutrophils likely has a significant, wide ranging clinical impact. PMID:26877061

  18. Study of baicalin on sympathoexcitation induced by myocardial ischemia via P2X3 receptor in superior cervical ganglia.

    PubMed

    Zhang, Jun; Liu, Shuangmei; Xu, Baohua; Li, Guodong; Li, Guilin; Huang, An; Wu, Bing; Peng, Lichao; Song, Miaomiao; Xie, Qiuyu; Lin, Weijian; Xie, Wei; Wen, Shiyao; Zhang, Zhedong; Xu, Xiaoling; Liang, Shangdong

    2015-05-01

    After the myocardial ischemia, injured myocardial tissues released large quantity of ATP, which activated P2X3 receptor in superior cervical ganglia and made the SCG postganglionic neurons excited. Excitatory of sympathetic postganglionic efferent neurons increased the blood pressure and heart rates, which aggravated the myocardial ischemic injury. Baicalin has anti-inflammatory and anti-oxidant properties. Our study showed that baicalin reduced the incremental concentration of serum CK-MB, cTn-T, epinephrine and ATP, decreased the up-regulated expression levels of P2X3 mRNA and protein in SCG after MI, and then inhibited the sympathetic excitatory activity triggered by MI injury. These results indicated that baicalin acted on P2X3 receptor was involved in the transmission of sympathetic excitation after the myocardial ischemic injury. Baicalin might decrease sympathetic activity via inhibiting P2X3 receptor in rat SCG to protect the myocardium. PMID:25554221

  19. Caspase-11 Requires the Pannexin-1 Channel and the Purinergic P2X7 Pore to Mediate Pyroptosis and Endotoxic Shock.

    PubMed

    Yang, Dahai; He, Yuan; Muñoz-Planillo, Raul; Liu, Qin; Núñez, Gabriel

    2015-11-17

    The noncanonical inflammasome induced by intracellular lipopolysaccharide (LPS) leads to caspase-11-dependent pyroptosis, which is critical for induction of endotoxic shock in mice. However, the signaling pathway downstream of caspase-11 is unknown. We found that cytosolic LPS stimulation induced caspase-11-dependent cleavage of the pannexin-1 channel followed up by ATP release, which in turn activated the purinergic P2X7 receptor to mediate cytotoxicity. In the absence of P2X7 or pannexin-1, pyroptosis induced by cytosolic LPS was abrogated. Cleavage of pannexin-1 required the catalytic activity of caspase-11 and was essential for ATP release and P2X7-mediated pyroptosis. Priming the caspase-11 pathway in vivo with LPS or Toll-like receptor-3 (TLR3) agonist resulted in high mortality in wild-type mice after secondary LPS challenge, but not in Casp11(-/-), Panx1(-/-), or P2x7(-/-) mice. These results reveal a critical role for pannexin-1 and P2X7 downstream of caspase-11 for pyroptosis and susceptibility to sepsis induced by the noncanonical inflammasome. PMID:26572062

  20. Caspase-11 requires the pannexin-1 channel and the purinergic P2X7 pore to mediate pyroptosis and endotoxic shock

    PubMed Central

    Yang, Dahai; He, Yuan; Muñoz-Planillo, Raul; Liu, Qin; Núñez, Gabriel

    2016-01-01

    SUMMARY The noncanonical inflammasome induced by intracellular lipopolysaccharide (LPS) leads to caspase-11-dependent pyroptosis which is critical for induction of endotoxic shock in mice. However, the signaling pathway downstream of caspase-11 is unknown. We found that cytosolic LPS stimulation induced caspase-11-dependent cleavage of the pannexin-1 channel and ATP release, which in turn activated the purinergic P2X7 receptor to mediate cytotoxicity. In the absence of P2X7 or pannexin-1, pyroptosis induced by LPS transfection or treatment with cholera toxin B and LPS was abrogated. Cleavage of pannexin-1 required the catalytic activity of caspase-11 and was essential for ATP release and P2X7-mediated pyroptosis. Priming the caspase-11 pathway in vivo with LPS or toll-like receptor-3 (TLR3) agonist resulted in high mortality in wild-type mice after secondary LPS challenge, but not in Casp11−/−, Panx1−/− or P2x7−/− mice. These results reveal a critical role for pannexin-1 and P2X7 downstream of caspase-11 for pyroptosis and susceptibility to sepsis induced by the noncanonical inflammasome. PMID:26572062

  1. P2X7 receptor-mediated killing of an intracellular parasite, Toxoplasma gondii, by human and murine macrophages1

    PubMed Central

    Lees, Michael P.; Fuller, Stephen J.; McLeod, Rima; Boulter, Nicola R.; Miller, Catherine M.; Zakrzewski, Alana M.; Mui, Ernest J.; Witola, William H.; Coyne, Jessica J.; Hargrave, Aubrey C.; Jamieson, Sarra E.; Blackwell, Jenefer M.; Wiley, James S.; Smith, Nicholas C.

    2010-01-01

    The P2X7 receptor (P2X7R)4 is highly expressed on the macrophage cell surface and activation of infected cells by extracellular ATP has been shown to kill intracellular bacteria and parasites. Furthermore, single nucleotide polymorphisms (SNPs) that decrease receptor function reduce the ability of human macrophages to kill Mycobacterium tuberculosis and are associated with extrapulmonary tuberculosis. In this paper we show that macrophages from people with the 1513C (rs3751143) loss-of-function P2X7R SNP are less effective in killing intracellular Toxoplasma gondii after exposure to ATP compared with macrophages from people with the 1513A wild-type allele. Supporting a P2X7R-specific effect on T. gondii, macrophages from P2X7R knock-out mice (P2X7R−/−) are unable to kill T. gondii as effectively as macrophages from wild-type mice. We show that P2X7R-mediated T. gondii killing occurs in parallel with host cell apoptosis and is independent of NO production. PMID:20488797

  2. Potent Suppressive Effects of 1-Piperidinylimidazole Based Novel P2X7 Receptor Antagonists on Cancer Cell Migration and Invasion.

    PubMed

    Park, Jin-Hee; Williams, Darren R; Lee, Ji-Hyung; Lee, So-Deok; Lee, Je-Heon; Ko, Hyojin; Lee, Ga-Eun; Kim, Sujin; Lee, Jeong-Min; Abdelrahman, Aliaa; Müller, Christa E; Jung, Da-Woon; Kim, Yong-Chul

    2016-08-25

    The P2X7 receptor (P2X7R) has been reported as a key mediator in inflammatory processes and cancer invasion/metastasis. In this study, we report the discovery of novel P2X7R antagonists and their functional activities as potential antimetastatic agents. Modifications of the hydantoin core-skeleton and the side chain substituents of the P2X7R antagonist 7 were performed. The structure-activity relationships (SAR) and optimization demonstrated the importance of the sulfonyl group at the R1 position and the substituted position and overall size of R2 for P2X7R antagonism. The optimized novel analogues displayed potent P2X7 receptor antagonism (IC50 = 0.11-112 nM) along with significant suppressive effects on IL-1β release (IC50 = 0.32-210 nM). Moreover, representative antagonists (12g, 13k, and 17d) with imidazole and uracil core skeletons significantly inhibited the invasion of MDA-MB-231 triple negative breast cancer cells and cancer cell migration in a zebrafish xenograft model, suggesting the potential therapeutic application of these novel P2X7 antagonists to block metastatic cancer. PMID:27427902

  3. Mechanistic insights from resolving ligand-dependent kinetics of conformational changes at ATP-gated P2X1R ion channels.

    PubMed

    Fryatt, Alistair G; Dayl, Sudad; Cullis, Paul M; Schmid, Ralf; Evans, Richard J

    2016-01-01

    Structural studies of P2X receptors show a novel U shaped ATP orientation following binding. We used voltage clamp fluorometry (VCF) and molecular dynamics (MD) simulations to investigate agonist action. For VCF the P2X1 receptor (P2X1R) K190C mutant (adjacent to the agonist binding pocket) was labelled with the fluorophore MTS-TAMRA and changes in fluorescence on agonist treatment provided a real time measure of conformational changes. Studies with heteromeric channels incorporating a key lysine mutation (K68A) in the ATP binding site demonstrate that normally three molecules of ATP activate the receptor. The time-course of VCF responses to ATP, 2'-deoxy ATP, 3'-deoxy ATP, Ap5A and αβmeATP were agonist dependent. Comparing the properties of the deoxy forms of ATP demonstrated the importance of the 2' hydroxyl group on the ribose ring in determining agonist efficacy consistent with MD simulations showing that it forms a hydrogen bond with the γ-phosphate oxygen stabilizing the U-shaped conformation. Comparison of the recovery of fluorescence on agonist washout, with channel activation to a second agonist application for the partial agonists Ap5A and αβmeATP, showed a complex relationship between conformational change and desensitization. These results highlight that different agonists induce distinct conformational changes, kinetics and recovery from desensitization at P2X1Rs. PMID:27616669

  4. Role of P2X7 Receptor in an Animal Model of Mania Induced by D-Amphetamine.

    PubMed

    Gubert, Carolina; Fries, Gabriel Rodrigo; Pfaffenseller, Bianca; Ferrari, Pâmela; Coutinho-Silva, Robson; Morrone, Fernanda Bueno; Kapczinski, Flávio; Battastini, Ana Maria Oliveira

    2016-01-01

    The objective of this study was to explore the association between the P2X7 purinergic receptor (P2X7R) and neuroinflammation using a preclinical model of acute bipolar mania. We analyzed the modulatory effects of P2X7R agonist (3'-O-(4-benzoyl)benzoyl-adenosine 5'-triphosphate, BzATP) and antagonists (brilliant blue, BBG and 3-[[5-(2,3 dichlorophenyl)-1H-tetrazol-1-yl]methyl]pyridine hydrochloride, A438079) on assessments related to behavior (locomotor activity), neuroinflammation (interleukin-1 beta, IL-1β; tumor necrosis factor alpha, TNF-α; and interleukin- 6, IL-6), oxidative stress (thiobarbituric acid reactive substances, TBARS) and neuroplasticity (brain-derived neurotrophic factor, BDNF) markers in a pharmacological model of mania induced by acute and chronic treatment with D-amphetamine (AMPH) (2 mg/kg) in mice. An apparent lack of responsiveness to AMPH was observed in terms of the locomotor activity in animals with blocked P2X7R or with genetic deletion of P2X7R in knockout (P2X7R(-/-)) mice. Likewise, P2X7R participated in the AMPH-induced increase of the proinflammatory and excitotoxic environment, as demonstrated by the reversal of IL-1β, TNF-α, and TBARS levels caused by P2X7R blocking. Our results support the hypothesis that P2X7R plays a role in the neuroinflammation induced by AMPH in a preclinical model of mania, which could explain the altered behavior. The present data suggest that P2X7R may be a therapeutic target related to the neuroinflammation reported in bipolar disorder. PMID:25502294

  5. Hypoxia induced amoeboid microglial cell activation in postnatal rat brain is mediated by ATP receptor P2X4

    PubMed Central

    2011-01-01

    Background Activation of amoeboid microglial cells (AMC) and its related inflammatory response have been linked to the periventricular white matter damage after hypoxia in neonatal brain. Hypoxia increases free ATP in the brain and then induces various effects through ATP receptors. The present study explored the possible mechanism in ATP induced AMC activation in hypoxia. Results We first examined the immunoexpression of P2X4, P2X7 and P2Y12 in the corpus callosum (CC) and subependyma associated with the lateral ventricles where both areas are rich in AMC. Among the three purinergic receptors, P2X4 was most intensely expressed. By double immunofluorescence, P2X4 was specifically localized in AMC (from P0 to P7) but the immunofluorescence in AMC was progressively diminished with advancing age (P14). It was further shown that P2X4 expression was noticeably enhanced in P0 day rats subjected to hypoxia and killed at 4, 24, 72 h and 7 d versus their matching controls by double labeling and western blotting analysis. P2X4 expression was most intense at 7 d whence the inflammatory response was drastic after hypoxia. We then studied the association of P2X4 with cytokine release in AMC after hypoxic exposure. In primary microglial cells exposed to hypoxia, IL-1β and TNF-α protein levels were up-regulated. Blockade of P2X4 receptor with 2', 3'-0-(2, 4, 6-Trinitrophenyl) adenosine 5'-triphosphate, a selective P2X1-7 blocker resulted in partial suppression of IL-1β (24% vs hypoxic group) and TNF-α expression (40% vs hypoxic group). However, pyridoxal phosphate-6-azo (benzene-2, 4-disulfonic acid) tetrasodium salt hydrate, a selective P2X1-3, 5-7 blocker did not exert any significant effect on the cytokine expression. Conclusions It is concluded that P2X4 which is constitutively expressed by AMC in postnatal rats was enhanced in hypoxia. Hypoxia induced increase in IL-1β and TNF-α expression was reversed by 2', 3'-0-(2, 4, 6-Trinitrophenyl) adenosine 5'-triphosphate

  6. The First CNS-Active Carborane: A Novel P2X7 Receptor Antagonist with Antidepressant Activity

    PubMed Central

    2014-01-01

    Relative to other polycyclic frameworks (1–3), a carborane cage (4 and Cs·5) exerts a significant biological effect as an inhibitor of the purinergic P2X7 receptor (P2X7R) which allows one to target depression in vivo and thus demonstrate, for the first time, that a carborane has the capacity to modify CNS activity. PMID:24689484

  7. P2X7 Receptor Activation Impairs Exogenous MHC Class I Oligopeptides Presentation in Antigen Presenting Cells

    PubMed Central

    Baroja-Mazo, Alberto; Barberà-Cremades, Maria; Pelegrín, Pablo

    2013-01-01

    Major histocompatibility complex class I (MHC I) on antigen presenting cells (APCs) is a potent molecule to activate CD8+ T cells and initiate immunity. P2X7 receptors (P2X7Rs) are present on the plasma membrane of APCs to sense the extracellular danger signal adenosine-5′-triphosphate (ATP). P2X7R activates the inflammasome and the release of IL-1β in macrophages and other immune cells to initiate the inflammatory response. Here we show that P2X7R stimulation by ATP in APCs decreased the amount of MHC I at the plasma membrane. Specific antagonism or genetic ablation of P2X7R inhibited the effects of ATP on levels of cellular MHC I. Furthermore, P2X7R stimulation was able to inhibit activation of CD8+ T cells via specific MHC I-oligopeptide complexes. Our study suggests that P2X7R activation on APCs is a novel inhibitor of adaptive CD8+ T cell immunity. PMID:23940597

  8. P2X7 receptor activation impairs exogenous MHC class I oligopeptides presentation in antigen presenting cells.

    PubMed

    Baroja-Mazo, Alberto; Barberà-Cremades, Maria; Pelegrín, Pablo

    2013-01-01

    Major histocompatibility complex class I (MHC I) on antigen presenting cells (APCs) is a potent molecule to activate CD8(+) T cells and initiate immunity. P2X7 receptors (P2X7Rs) are present on the plasma membrane of APCs to sense the extracellular danger signal adenosine-5'-triphosphate (ATP). P2X7R activates the inflammasome and the release of IL-1β in macrophages and other immune cells to initiate the inflammatory response. Here we show that P2X7R stimulation by ATP in APCs decreased the amount of MHC I at the plasma membrane. Specific antagonism or genetic ablation of P2X7R inhibited the effects of ATP on levels of cellular MHC I. Furthermore, P2X7R stimulation was able to inhibit activation of CD8(+) T cells via specific MHC I-oligopeptide complexes. Our study suggests that P2X7R activation on APCs is a novel inhibitor of adaptive CD8(+) T cell immunity. PMID:23940597

  9. P2X7 receptor-mediated calcium dynamics in HEK293 cells: experimental characterization and modelling approach

    NASA Astrophysics Data System (ADS)

    Di Garbo, A.; Alloisio, S.; Nobile, M.

    2012-04-01

    The P2X7 receptor (P2X7R) induces ionotropic Ca2 + signalling in different cell types. It plays an important role in the immune response and in the nervous system. Here, the mechanisms underlying intracellular Ca2 + variations evoked by 3‧-O-(4-benzoyl)benzoyl-ATP (BzATP), a potent agonist of the P2X7R, in transfected HEK293 cells, are investigated both experimentally and theoretically. We propose a minimal model of P2X7R that is capable of reproducing, qualitatively and quantitatively, the experimental data. This approach was also adopted for the P2X7R variant, which lacks the entire C-terminus tail (trP2X7R). Then we introduce a biophysical model describing the Ca2 + dynamics in HEK293. Our model gives an account of the ionotropic Ca2 + influx evoked by BzATP on the basis of the kinetics model of P2X7R. To explain the complex Ca2 + responses evoked by BzATP, the model predicted that an impairment in Ca2 + extrusion flux through the plasma membrane is a key factor for Ca2 + homeostasis in HEK293 cells.

  10. Synthesis, SAR, and Pharmacological Characterization of Brain Penetrant P2X7 Receptor Antagonists

    PubMed Central

    2015-01-01

    We describe the synthesis and SAR of 1,2,3-triazolopiperidines as a novel series of potent, brain penetrant P2X7 antagonists. Initial efforts yielded a series of potent human P2X7R antagonists with moderate to weak rodent potency, some CYP inhibition, poor metabolic stability, and low solubility. Further work in this series, which focused on the SAR of the N-linked heterocycle, not only increased the potency at the human P2X7R but also provided compounds with good potency at the rat P2X7R. These efforts eventually delivered a potent rat and human P2X7R antagonist with good physicochemical properties, an excellent pharmacokinetic profile, good partitioning into the CNS, and demonstrated in vivo target engagement after oral dosing. PMID:26101572

  11. Spontaneous cell fusion in macrophage cultures expressing high levels of the P2Z/P2X7 receptor.

    PubMed

    Chiozzi, P; Sanz, J M; Ferrari, D; Falzoni, S; Aleotti, A; Buell, G N; Collo, G; Di Virgilio, F

    1997-08-11

    Mouse and human macrophages express a plasma membrane receptor for extracellular ATP named P2Z/P2X7. This molecule, recently cloned, is endowed with the intriguing property of forming an aqueous pore that allows transmembrane fluxes of hydrophylic molecules of molecular weight below 900. The physiological function of this receptor is unknown. In a previous study we reported experiments suggesting that the P2Z/P2X7 receptor is involved in the formation of macrophage-derived multinucleated giant cells (MGCs; Falzoni, S., M. Munerati, D. Ferrari, S. Spisani, S. Moretti, and F. Di Virgilio. 1995. J. Clin. Invest. 95:1207- 1216). We have selected several clones of mouse J774 macrophages that are characterized by either high or low expression of the P2Z/P2X7 receptor and named these clones P2Zhyper or P2Zhypo, respectively. P2Zhyper, but not P2Zhypo, cells grown to confluence in culture spontaneously fuse to form MGCs. As previously shown for human macrophages, fusion is inhibited by the P2Z/P2X7 blocker oxidized ATP. MGCs die shortly after fusion through a dramatic process of cytoplasmic sepimentation followed by fragmentation. These observations support our previous hypothesis that the P2Z/P2X7 receptor is involved in macrophage fusion. PMID:9245796

  12. Association of IFN-γ and P2X7 Receptor Gene Polymorphisms in Susceptibility to Tuberculosis Among Iranian Patients.

    PubMed

    Shamsi, Mahdi; Zolfaghari, Mohammad Reza; Farnia, Parissa

    2016-03-01

    Interferon-gamma (IFN-γ) and P2X7 receptor are crucial for host defence against mycobacterial infections. Recent studies have indicated that IFN-γ, IFN-γ receptor 1 (IFN-γR1) andP2X7 gene polymorphisms are associated with susceptibility to pulmonary tuberculosis (TB). However, the relationship between IFN-γ and P2X7 polymorphism and TB susceptibility remains inconclusive in Iranian population. For this reason, single nucleotide polymorphisms (SNPs) in IFN-γ (G+2109A), IFN-γR1 (G-611A) and P2X7 genes (at -762, 1513 position) in patients (n = 100) were assessed using PCR-RFLP. Data were analysed with SPSS version 18. For the 2109 loci of IFN-γ gene, the frequency of mutant alleles between patients and controls were not statistically significant. However, there was a significant difference between the TB patient and controls for -611 alleles of IFN-γR1 (P = 0.01). Additionally, the frequency of P2X7 gene polymorphisms (SNP-762 and 1513) between patients and controls was statistically significant. In conclusions, our study revealed a significant association of IFN-γR1 and P2X7 genes polymorphisms with risk of developing TB in Iranian population. PMID:27020872

  13. Regulation of the desensitization and ion selectivity of ATP-gated P2X2 channels by phosphoinositides.

    PubMed

    Fujiwara, Yuichiro; Kubo, Yoshihiro

    2006-10-01

    Phosphoinositides (PIP(n)s) are known to regulate the activity of some ion channels. Here we determined that ATP-gated P2X(2) channels also are regulated by PIP(n)s, and investigated the structural background and the unique features of this regulation. We initially used two-electrode voltage clamp to analyse the electrophysiological properties of P2X(2) channels expressed in Xenopus oocytes, and observed that preincubation with wortmannin or LY294002, two PI3K inhibitors, accelerated channel desensitization. K365Q or K369Q mutation of the conserved, positively charged, amino acid residues in the proximal region of the cytoplasmic C-terminal domain also accelerated desensitization, whereas a K365R or K369R mutation did not. We observed that the permeability of the channel to N-methyl-d-glucamine (NMDG) transiently increased and then decreased after ATP application, and that the speed of the decrease was accelerated by K365Q or K369Q mutation or PI3K inhibition. Using GST-tagged recombinant proteins spanning the proximal C-terminal region, we then analysed their binding of the P2X(2) cytoplasmic domain to anionic lipids using PIP(n)s-coated nitrocellulose membranes. We found that the recombinant proteins that included the positively charged region bound to PIPs and PIP(2)s, and that this binding was eliminated by the K365Q and K369Q mutations. We also used a fluorescence assay to confirm that fusion proteins comprising the proximal C-terminal region of P2X(2) with EGFP expressed in COS-7 cells closely associated with the membrane. Taken together, these results show that membrane-bound PIP(n)s play a key role in maintaining channel activity and regulating pore dilation through electrostatic interaction with the proximal region of the P2X(2) cytoplasmic C-terminal domain. PMID:16857707

  14. New P2X3 receptor antagonists. Part 2: Identification and SAR of quinazolinones.

    PubMed

    Szántó, Gábor; Makó, Attila; Vágó, István; Hergert, Tamás; Bata, Imre; Farkas, Bence; Kolok, Sándor; Vastag, Mónika

    2016-08-15

    Numerous potent P2X3 antagonists have been discovered and the therapeutic potential of P2X3 antagonism already comprises proof-of-concept data obtained in clinical trials with the most advanced compound. We have lately reported the discovery and optimization of thia-triaza-tricycle compounds with potent P2X3 antagonistic properties. This Letter describes the SAR of a back-up series containing a 4-oxo-quinazoline central ring. The discovery of the highly potent compounds 51 is presented. PMID:27426300

  15. B-Type Natriuretic Peptide-Induced Delayed Modulation of TRPV1 and P2X3 Receptors of Mouse Trigeminal Sensory Neurons

    PubMed Central

    Ntamati, Niels; Nistri, Andrea

    2013-01-01

    Important pain transducers of noxious stimuli are small- and medium-diameter sensory neurons that express transient receptor vanilloid-1 (TRPV1) channels and/or adenosine triphosphate (ATP)-gated P2X3 receptors whose activity is upregulated by endogenous neuropeptides in acute and chronic pain models. Little is known about the role of endogenous modulators in restraining the expression and function of TRPV1 and P2X3 receptors. In dorsal root ganglia, evidence supports the involvement of the natriuretic peptide system in the modulation of nociceptive transmission especially via the B-type natriuretic peptide (BNP) that activates the natriuretic peptide receptor-A (NPR-A) to downregulate sensory neuron excitability. Since the role of BNP in trigeminal ganglia (TG) is unclear, we investigated the expression of BNP in mouse TG in situ or in primary cultures and its effect on P2X3 and TRPV1 receptors of patch-clamped cultured neurons. Against scant expression of BNP, almost all neurons expressed NPR-A at membrane level. While BNP rapidly increased cGMP production and Akt kinase phosphorylation, there was no early change in passive neuronal properties or responses to capsaicin, α,β-meATP or GABA. Nonetheless, 24 h application of BNP depressed TRPV1 mediated currents (an effect blocked by the NPR-A antagonist anantin) without changing responses to α,β-meATP or GABA. Anantin alone decreased basal cGMP production and enhanced control α,β-meATP-evoked responses, implying constitutive regulation of P2X3 receptors by ambient BNP. These data suggest a slow modulatory action by BNP on TRPV1 and P2X3 receptors outlining the role of this peptide as a negative regulator of trigeminal sensory neuron excitability to nociceptive stimuli. PMID:24312267

  16. Silencing P2X7 receptor downregulates the expression of TCP-1 involved in lymphoma lymphatic metastasis

    PubMed Central

    Yang, Ziyi; Zeng, Jia; Zhang, Yi; Song, Yang; Kong, Ying; Ren, Shuangyi; Zuo, Yunfei

    2015-01-01

    P2X7R is an ATP-gated cation channel that participates in cell proliferation and apoptosis. TCP-1 assists with the protein folding. According to our previous research, the P2X7R has a potential role in P388D1 lymphoid neoplasm cells dissemination to peripheral lymph nodes. In order to make a further exploration about the probable mechanism, the lymph nodes which metastasized by P2X7R-silenced P388D1 cells or non-silenced cells were analyzed by 2DE and a MALDI-TOF-based proteomics approach. In the 64 proteins which were differentially expressed between two groups, TCP-1 was found to be significantly decreased in P2X7R shRNA group compared to controls. This correlation was also found in subsequent experiments in vivo and in vitro. The positive correlation between P2X7R and TCP-1 was also proved in both lymphoma and benign lymphadenopathy tissues from patients. It indicates that TCP-1 may be a crucial downstream molecular of P2X7R and plays a novel role in lymphoid neoplasm metastasis. PMID:26556873

  17. A novel P2X4 receptor-selective antagonist produces anti-allodynic effect in a mouse model of herpetic pain.

    PubMed

    Matsumura, Yuta; Yamashita, Tomohiro; Sasaki, Atsushi; Nakata, Eriko; Kohno, Keita; Masuda, Takahiro; Tozaki-Saitoh, Hidetoshi; Imai, Toshiyasu; Kuraishi, Yasushi; Tsuda, Makoto; Inoue, Kazuhide

    2016-01-01

    Accumulating evidence indicates that purinergic P2X4 receptors (P2X4R: cation channels activated by extracellular ATP) expressed in spinal microglia are crucial for pathological chronic pain caused by nerve damage, suggesting a potential target for drug discovery. We identified NP-1815-PX (5-[3-(5-thioxo-4H-[1,2,4]oxadiazol-3-yl)phenyl]-1H-naphtho[1, 2-b][1,4]diazepine-2,4(3H,5H)-dione) as a novel antagonist selective for P2X4R with high potency and selectivity compared with other P2XR subtypes. In in vivo assay for acute and chronic pain, intrathecal administration of NP-1815-PX produced an anti-allodynic effect in mice with traumatic nerve damage without affecting acute nociceptive pain and motor function (although its oral administration did not produce the effect). Furthermore, in a mouse model of herpetic pain, P2X4R upregulation in the spinal cord exclusively occurred in microglia, and intrathecal NP-1815-PX suppressed induction of mechanical allodynia. This model also showed K(+)/Cl(-) cotransporter 2 (KCC2) downregulation, which is implicated in dorsal horn neuron hyperexcitability; this downregulation was restored by intrathecal treatment with NP-1815-PX or by interfering with brain-derived neurotrophic factor (BDNF) signaling, a P2X4R-activated microglial factor implicated in KCC2 downregulation. Taken together, the newly developed P2X4R antagonist NP-1815-PX produces anti-allodynic effects in chronic pain models without altering acute pain sensitivity, suggesting that microglial P2X4R could be an attractive target for treating chronic pain. PMID:27576299

  18. A novel P2X4 receptor-selective antagonist produces anti-allodynic effect in a mouse model of herpetic pain

    PubMed Central

    Matsumura, Yuta; Yamashita, Tomohiro; Sasaki, Atsushi; Nakata, Eriko; Kohno, Keita; Masuda, Takahiro; Tozaki-Saitoh, Hidetoshi; Imai, Toshiyasu; Kuraishi, Yasushi; Tsuda, Makoto; Inoue, Kazuhide

    2016-01-01

    Accumulating evidence indicates that purinergic P2X4 receptors (P2X4R: cation channels activated by extracellular ATP) expressed in spinal microglia are crucial for pathological chronic pain caused by nerve damage, suggesting a potential target for drug discovery. We identified NP-1815-PX (5-[3-(5-thioxo-4H-[1,2,4]oxadiazol-3-yl)phenyl]-1H-naphtho[1, 2-b][1,4]diazepine-2,4(3H,5H)-dione) as a novel antagonist selective for P2X4R with high potency and selectivity compared with other P2XR subtypes. In in vivo assay for acute and chronic pain, intrathecal administration of NP-1815-PX produced an anti-allodynic effect in mice with traumatic nerve damage without affecting acute nociceptive pain and motor function (although its oral administration did not produce the effect). Furthermore, in a mouse model of herpetic pain, P2X4R upregulation in the spinal cord exclusively occurred in microglia, and intrathecal NP-1815-PX suppressed induction of mechanical allodynia. This model also showed K+/Cl− cotransporter 2 (KCC2) downregulation, which is implicated in dorsal horn neuron hyperexcitability; this downregulation was restored by intrathecal treatment with NP-1815-PX or by interfering with brain-derived neurotrophic factor (BDNF) signaling, a P2X4R-activated microglial factor implicated in KCC2 downregulation. Taken together, the newly developed P2X4R antagonist NP-1815-PX produces anti-allodynic effects in chronic pain models without altering acute pain sensitivity, suggesting that microglial P2X4R could be an attractive target for treating chronic pain. PMID:27576299

  19. Purinergic Autocrine Regulation of Mechanosensitivity and Serotonin Release in a Human EC Model: ATP-gated P2X3 Channels in EC are Downregulated in Ulcerative Colitis

    PubMed Central

    Liñán-Rico, Andrómeda; Wunderlich, Jacqueline E.; Grants, Iveta S.; Frankel, Wendy L.; Xue, Jianjing; Williams, Kent C.; Harzman, Alan E.; Enneking, Joshua T.; Cooke, Helen J.; Christofi, Fievos L.

    2014-01-01

    Background Alterations in 5-hydroxytryptamine (HT) signaling in inflamed gut may contribute to pathogenesis of inflammatory bowel diseases. Adenosine 5′-triphosphate (ATP) regulates mucosal-mechanosensory reflexes and ATP receptors are sensitive to mucosal inflammation. Yet, it remains unknown whether ATP can modulate 5-HT signaling in enterochromaffin cells (EC). We tested the novel purinergic hypothesis that ATP is a critical autocrine regulator of EC mechanosensitivity and whether EC expression of ATP-gated P2X3-ion channels is altered in inflammatory bowel diseases. Methods Laser confocal (fluo-4) Ca2+ imaging was performed in 1947 BON cells. Chemical stimulation or mechanical stimulation (MS) was used to study 5-HT or ATP release in human BON or surgical mucosal specimens, and purine receptors by reverse transcription-polymerase chain reaction, Western Blot, or P2X3-immunoreactivity in BON or 5-HT+ human EC (hEC) in 11 control and 10 severely inflamed ulcerative colitis (UC) cases. Results ATP or MS triggered Ca2+-transients or 5-HT release in BON. ATP or adenosine diphosphate increased 5-HT release 5-fold. MS caused ATP release, detected after 5′ecto-ATPase inhibition by ARL67156. ARL67156 augmented and apyrase blocked Ca2+/5-HT mechanosensitive responses. 2-Methyl-thio-adenosine diphosphate 5′-monophosphate-evoked (P2Y1,12) or mechanically-evoked responses were blocked or augmented by a P2Y1,12 antagonist, MRS2179, in different cells or inhibited by U73122. A P2Y12 antagonist, 2MeSAMP, augmented responses. A P2X1,3 agonist, α,β-MeATP, triggered Ca2+ responses, whereas a P2X1,2/3,3 antagonist, 2′,3′-O-(2,4,6-trinitrophenyl)-ATP, blocked mechanical responses or cell-surface 5′ATP-TR labeling. In hEC, α,β-MeATP stimulated 5-HT release. In UC, P2X3-immunoreactivity decreased from 15% to 0.2% of 5-HT+hECs. Human mucosa and BON expressed P2X1, P2X3, P2X4, P2X5, P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, and P2Y12R-messenger RNA transcripts. Conclusions ATP is

  20. Combined, but not individual, blockade of ASIC3, P2X, and EP4 receptors attenuates the exercise pressor reflex in rats with freely perfused hindlimb muscles.

    PubMed

    Stone, Audrey J; Copp, Steven W; Kim, Joyce S; Kaufman, Marc P

    2015-12-01

    In healthy humans, tests of the hypothesis that lactic acid, PGE2, or ATP plays a role in evoking the exercise pressor reflex proved controversial. The findings in humans resembled ours in decerebrate rats that individual blockade of the receptors to lactic acid, PGE2, and ATP had only small effects on the exercise pressor reflex provided that the muscles were freely perfused. This similarity between humans and rats prompted us to test the hypothesis that in rats with freely perfused muscles combined receptor blockade is required to attenuate the exercise pressor reflex. We first compared the reflex before and after injecting either PPADS (10 mg/kg), a P2X receptor antagonist, APETx2 (100 μg/kg), an activating acid-sensing ion channel 3 (ASIC) channel antagonist, or L161982 (2 μg/kg), an EP4 receptor antagonist, into the arterial supply of the hindlimb of decerebrated rats. We then examined the effects of combined blockade of P2X receptors, ASIC3 channels, and EP4 receptors on the exercise pressor reflex using the same doses, intra-arterial route, and time course of antagonist injections as those used for individual blockade. We found that neither PPADS (n = 5), APETx2 (n = 6), nor L161982 (n = 6) attenuated the reflex. In contrast, combined blockade of these receptors (n = 7) attenuated the peak (↓27%, P < 0.019) and integrated (↓48%, P < 0.004) pressor components of the reflex. Combined blockade injected intravenously had no effect on the reflex. We conclude that combined blockade of P2X receptors, ASIC3 channels, and EP4 receptors on the endings of thin fiber muscle afferents is required to attenuate the exercise pressor reflex in rats with freely perfused hindlimbs. PMID:26472871

  1. Loss of inhibition by brain natriuretic peptide over P2X3 receptors contributes to enhanced spike firing of trigeminal ganglion neurons in a mouse model of familial hemiplegic migraine type-1.

    PubMed

    Marchenkova, Anna; van den Maagdenberg, Arn M J M; Nistri, Andrea

    2016-09-01

    Purinergic P2X3 receptors (P2X3Rs) play an important role in pain pathologies, including migraine. In trigeminal neurons, P2X3Rs are constitutively downregulated by endogenous brain natriuretic peptide (BNP). In a mouse knock-in (KI) model of familial hemiplegic migraine type-1 with upregulated calcium CaV2.1 channel function, trigeminal neurons exhibit hyperexcitability with gain-of-function of P2X3Rs and their deficient BNP-mediated inhibition. We studied whether the absent BNP-induced control over P2X3Rs activity in KI cultures may be functionally expressed in altered firing activity of KI trigeminal neurons. Patch-clamp experiments investigated the excitability of wild-type and KI trigeminal neurons induced by either current or agonists for P2X3Rs or transient receptor potential vanilloid-1 (TRPV1) receptors. Consistent with the constitutive inhibition of P2X3Rs by BNP, sustained pharmacological block of BNP receptors selectively enhanced P2X3R-mediated excitability of wild-type neurons without affecting firing evoked by the other protocols. This effect included increased number of action potentials, lower spike threshold and shift of the firing pattern distribution toward higher spiking activity. Thus, inactivation of BNP signaling transformed the wild-type excitability phenotype into the one typical for KI. BNP receptor block did not influence excitability of KI neurons in accordance with the lack of BNP-induced P2X3R modulation. Our study suggests that, in wild-type trigeminal neurons, negative control over P2X3Rs by the BNP pathway is translated into tonic suppression of P2X3Rs-mediated excitability. Lack of this inhibition in KI cultures results in a hyperexcitability phenotype and might contribute to facilitated trigeminal pain transduction relevant for migraine. PMID:27346147

  2. P2X7 receptor activation in rat brain cultured astrocytes increases the biosynthetic release of cysteinyl leukotrienes.

    PubMed

    Ballerini, P; Ciccarelli, R; Caciagli, F; Rathbone, M P; Werstiuk, E S; Traversa, U; Buccella, S; Giuliani, P; Jang, S; Nargi, E; Visini, D; Santavenere, C; Di Iorio, P

    2005-01-01

    Astrocytes have been recognized as important elements in controlling inflammatory as well as immune processes in the central nervous system (CNS). Recently, glial cells have been shown to produce cysteinyl leukotrienes (CysLTs) which are known lipid mediators of inflammation and whose extracellular concentrations rise under different pathological conditions in the brain. In the same conditions also extracellular concentrations of ATP dramatically increase reaching levels able to activate P2X7 ionotropic receptors for which an emerging role in neuroinflammation and neurodegeneration has been claimed. RTPCR analysis showed that primary cultures of rat brain astrocytes express P2X7 receptors. Application of the selective P2X7 agonist benzoyl benzoly ATP (BzATP) markedly increased [Ca2+]i which was mediated by a calcium influx from the extracellular milieu. The P2X7 antagonist, oATP, suppressed the BzATP-induced calcium increase. Consistent with the evidence that increased calcium levels activate the leukotriene biosynthetic pathway, challenge of astrocytes with either the calcium ionophore A23187 or BzATP significantly increased CysLT production and the cell pre-treatment with EGTA abolished these effects. Again the P2X7 antagonist prevented the BzATP-mediated CysLT efflux, whereas the astrocyte pretreatment with MK-571, a CysLT1 receptor antagonist, was ineffective. The astrocyte pre-treatment with a cocktail of inhibitors of ATP binding cassette (ABC) proteins reduced the BzATP-mediated CysLT production confirming that ABC transporters are involved in the release of CysLTs. The astrocyte P2X7- evoked rise of CysLT efflux was abolished in the presence of MK-886, an inhibitor of 5-lipoxygenase activating protein (FLAP) whose expression, along with that of 5-lipoxygenase (5-LO) was reported by Northern Blot analysis. The stimulation of P2X7 induced an up-regulation of FLAPmRNA that was reduced by the antagonist oATP. These data suggest that in rat brain cultured

  3. Characterization of P2X3, P2Y1 and P2Y4 receptors in cultured HEK293-hP2X3 cells and their inhibition by ethanol and trichloroethanol.

    PubMed

    Fischer, Wolfgang; Wirkner, Kerstin; Weber, Marco; Eberts, Christoph; Köles, Laszlo; Reinhardt, Robert; Franke, Heike; Allgaier, Clemens; Gillen, Clemens; Illes, Peter

    2003-05-01

    Membrane currents and changes in the intracellular Ca2+ concentration ([Ca2+]i) were measured in HEK293 cells transfected with the human P2X3 receptor (HEK293-hP2X3). RT-PCR and immunocytochemistry indicated the additional presence of endogenous P2Y1 and to some extent P2Y4 receptors. P2 receptor agonists induced inward currents in HEK293-hP2X3 cells with the rank order of potency alpha,beta-meATP approximately ATP > ADP-beta-S > UTP. A comparable rise in [Ca2+]i was observed after the slow superfusion of ATP, ADP-beta-S and UTP; alpha,beta-meATP was ineffective. These data, in conjunction with results obtained by using the P2 receptor antagonists TNP-ATP, PPADS and MRS2179 indicate that the current response to alpha,beta-meATP is due to P2X3 receptor activation, while the ATP-induced rise in [Ca2+]i is evoked by P2Y1 and P2Y4 receptor activation. TCE depressed the alpha,beta-meATP current in a manner compatible with a non-competitive antagonism. The ATP-induced increase of [Ca2+]i was much less sensitive to the inhibitory effect of TCE than the current response to alpha,beta-meATP. The present study indicates that in HEK293-hP2X3 cells, TCE, but not ethanol, potently inhibits ligand-gated P2X3 receptors and, in addition, moderately interferes with G protein-coupled P2Y1 and P2Y4 receptors. Such an effect may be relevant for the interruption of pain transmission in dorsal root ganglion neurons following ingestion of chloral hydrate or trichloroethylene. PMID:12694404

  4. Graft-Infiltrating Macrophages Adopt an M2 Phenotype and Are Inhibited by Purinergic Receptor P2X7 Antagonist in Chronic Rejection.

    PubMed

    Wu, C; Zhao, Y; Xiao, X; Fan, Y; Kloc, M; Liu, W; Ghobrial, R M; Lan, P; He, X; Li, X C

    2016-09-01

    Macrophages exhibit diverse phenotypes and functions; they are also a major cell type infiltrating chronically rejected allografts. The exact phenotypes and roles of macrophages in chronic graft loss remain poorly defined. In the present study, we used a mouse heart transplant model to examine macrophages in chronic allograft rejection. We found that treatment of C57BL/6 mice with CTLA4 immunoglobulin fusion protein (CTLA4-Ig) prevented acute rejection of a Balb/c heart allograft but allowed chronic rejection to develop over time, characterized by prominent neointima formation in the graft. There was extensive macrophage infiltration in the chronically rejected allografts, and the graft-infiltrating macrophages expressed markers associated with M2 cells but not M1 cells. In an in vitro system in which macrophages were polarized into either M1 or M2 cells, we screened phenotypic differences between M1 and M2 cells and identified purinergic receptor P2X7 (P2x7r), an adenosine triphosphate (ATP)-gated ion channel protein that was preferentially expressed by M2 cells. We further showed that blocking the P2x7r using oxidized ATP (oATP) inhibited M2 induction in a dose-dependent fashion in vitro. Moreover, treatment of C57BL/6 recipients with the P2x7r antagonist oATP, in addition to CTLA4-Ig treatment, inhibited graft-infiltrating M2 cells, prevented transplant vasculopathy, and induced long-term heart allografts survival. These findings highlight the importance of the P2x7r-M2 axis in chronic rejection and establish P2x7r as a potential therapeutic target in suppression of chronic rejection. PMID:27575724

  5. Extracellular ATP dissociates nonmuscle myosin from P2X(7) complex: this dissociation regulates P2X(7) pore formation.

    PubMed

    Gu, Ben J; Rathsam, Catherine; Stokes, Leanne; McGeachie, Andrew B; Wiley, James S

    2009-08-01

    The P2X(7) receptor is a ligand-gated cation channel that is highly expressed on monocyte-macrophages and that mediates the pro-inflammatory effects of extracellular ATP. Dilation of the P2X(7) channel and massive K(+) efflux follows initial channel opening, but the mechanism of secondary pore formation is unclear. The proteins associated with P2X(7) were isolated by using anti-P2X(7) monoclonal antibody-coated Dynabeads from both interferon-gamma plus LPS-stimulated monocytic THP-1 cells and P2X(7)-transfected HEK-293 cells. Two nonmuscle myosins, NMMHC-IIA and myosin Va, were found to associate with P2X(7) in THP-1 cells and HEK-293 cells, respectively. Activation of the P2X(7) receptor by ATP caused dissociation of P2X(7) from nonmuscle myosin in both cell types. The interaction of P2X(7) and NMMHC-IIA molecules was confirmed by fluorescent life time measurements and fluorescent resonance of energy transfer-based time-resolved flow cytometry assay. Reducing the expression of NMMHC-IIA or myosin Va by small interfering RNA or short hairpin RNA led to a significant increase of P2X(7) pore function without any increase in surface expression or ion channel function of P2X(7) receptors. S-l-blebbistatin, a specific inhibitor of NMMHC-IIA ATPase, inhibited both ATP-induced ethidium uptake and ATP-induced dissociation of P2X(7)-NMMHC-IIA complex. In both cell types nonmuscle myosin closely interacts with P2X(7) and is dissociated from the complex by extracellular ATP. Dissociation of this anchoring protein may be required for the transition of P2X(7) channel to a pore. PMID:19494237

  6. P2X and NMDA receptor involvement in temporomandibular joint-evoked reflex activity in rat jaw muscles.

    PubMed

    Watanabe, T; Tsuboi, Y; Sessle, B J; Iwata, K; Hu, J W

    2010-07-30

    We have previously shown that injection of the excitatory amino glutamate into the rat temporomandibular joint (TMJ) evokes reflex activity in both anterior digastric (DIG) and masseter (MASS) muscles that can be attenuated by prior TMJ injection of an N-methyl-d-aspartate (NMDA) receptor antagonist. The aim of the present study was to test if jaw muscle activity could also be evoked by P2X receptor agonist injection into the rat TMJ region and if the reflex activity could be modulated by TMJ injection of P2X receptor antagonist or NMDA receptor antagonist. The selective P2X subtype agonist alpha,beta-methylene adenosine 5'-triphosphate (alpha,beta-me ATP) and vehicle (phosphate-buffered saline) or the selective P2X antagonist, 2'-(or-3')-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate (TNP-ATP) or the selective NMDA antagonist (+/-)-d-2-amino-5-phosphonovalerate(APV) were injected into the rat TMJ region. Electromyographic (EMG) reflex activity was recorded in both DIG and MASS muscles. Compared with the baseline EMG activity, alpha,beta-me-ATP injection into the TMJ (but not its systemic administration) following pre-injection of the vehicle significantly increased the magnitude and the duration of ipsilateral DIG and MASS EMG activity in a dose-dependent manner. The alpha,beta-me-ATP-evoked responses could be antagonized by pre-injection of TNP-ATP into the same TMJ site but contralateral TMJ injection of TNP-ATP proved ineffective. Furthermore, the alpha,beta-me-ATP-evoked responses could also be antagonized by APV injected into the same TMJ site but not by its systemic injection. These results indicate the interaction of peripheral purinergic as well as glutamatergic receptor mechanisms in the processing of TMJ nociceptive afferent inputs that evoke reflex activity in jaw muscles. PMID:20501327

  7. Extracellular ATP induces intracellular alpha-synuclein accumulation via P2X1 receptor-mediated lysosomal dysfunction

    PubMed Central

    Gan, Ming; Moussaud, Simon; Jiang, Peizhou; McLean, Pamela J.

    2014-01-01

    The pathological hallmark of Parkinson’s disease (PD) is the accumulation of alpha-synuclein (αsyn) in susceptible neurons in the form of Lewy bodies and Lewy neurites. The etiology of PD remains unclear. Because brain injury has been suggested to facilitate αsyn aggregation, we investigated whether cellular breakdown products from damaged cells can act on neighboring healthy cells and cause intracellular αsyn accumulation/aggregation. Using two neuronal cell models we found that extracellular ATP induced a significant increase in intracellular αsyn levels between 24 to 48 hours after treatment. Further investigation revealed that the observed αsyn accumulation is a result of lysosome dysfunction caused by extracellular ATP-induced elevation of lysosomal pH. Interestingly, P2X1 receptor appears to mediate the cells’ response to extracellular ATP. Although Ca2+ influx via P2X1 receptor is necessary for αsyn accumulation, Ca2+ influx per se is not sufficient for increased αsyn accumulation. These findings provide new insight into our knowledge of the role of P2X receptors in PD pathogenesis and may be helpful in identifying new therapeutic targets for PD. PMID:25480524

  8. Extracellular ATP induces intracellular alpha-synuclein accumulation via P2X1 receptor-mediated lysosomal dysfunction.

    PubMed

    Gan, Ming; Moussaud, Simon; Jiang, Peizhou; McLean, Pamela J

    2015-02-01

    The pathologic hallmark of Parkinson's disease (PD) is the accumulation of alpha-synuclein (αsyn) in susceptible neurons in the form of Lewy bodies and Lewy neurites. The etiology of PD remains unclear. Because brain injury has been suggested to facilitate αsyn aggregation, we investigated whether cellular breakdown products from damaged cells can act on neighboring healthy cells and cause intracellular αsyn accumulation and/or aggregation. Using 2 neuronal cell models, we found that extracellular adenosine triphosphate (ATP) induced a significant increase in intracellular αsyn levels between 24 and 48 hours after treatment. Further investigation revealed that the observed αsyn accumulation is a result of lysosome dysfunction caused by extracellular ATP-induced elevation of lysosomal pH. Interestingly, P2X1 receptor appears to mediate the cells' response to extracellular ATP. Although Ca(2+) influx via P2X1 receptor is necessary for αsyn accumulation, Ca(2+) influx per se is not sufficient for increased αsyn accumulation. These findings provide new insight into our knowledge of the role of P2X receptors in PD pathogenesis and may be helpful in identifying new therapeutic targets for PD. PMID:25480524

  9. The therapeutic promise of ATP antagonism at P2X3 receptors in respiratory and urological disorders.

    PubMed

    Ford, Anthony P; Undem, Bradley J

    2013-01-01

    A sensory role for ATP was proposed long before general acceptance of its extracellular role. ATP activates and sensitizes signal transmission at multiple sites along the sensory axis, across multiple synapses. P2X and P2Y receptors mediate ATP modulation of sensory pathways and participate in dysregulation, where ATP action directly on primary afferent neurons (PANs), linking receptive field to CNS, has received much attention. Many PANs, especially C-fibers, are activated by ATP, via P2X3-containing trimers. P2X3 knock-out mice and knock-down in rats led to reduced nocifensive activity and visceral reflexes, suggesting that antagonism may offer benefit in sensory disorders. Recently, drug-like P2X3 antagonists, active in a many inflammatory and visceral pain models, have emerged. Significantly, these compounds have no overt CNS action and are inactive versus acute nociception. Selectively targeting ATP sensitization of PANs may lead to therapies that block inappropriate chronic signals at their source, decreasing drivers of peripheral and central wind-up, yet leaving defensive nociceptive and brain functions unperturbed. This article reviews this evidence, focusing on how ATP sensitization of PANs in visceral "hollow" organs primes them to chronic discomfort, irritation and pain (symptoms) as well as exacerbated autonomic reflexes (signs), and how the use of isolated organ-nerve preparations has revealed this mechanism. Urinary and airways systems share many features: dependence on continuous afferent traffic to brainstem centers to coordinate efferent autonomic outflow; loss of descending inhibitory influence in functional and sensory disorders; dependence on ATP in mediating sensory responses to diverse mechanical and chemical stimuli; a mechanistically overlapping array of existing medicines for pathological conditions. These similarities may also play out in terms of future treatment of signs and symptoms, in the potential for benefit of P2X3 antagonists

  10. The therapeutic promise of ATP antagonism at P2X3 receptors in respiratory and urological disorders

    PubMed Central

    Ford, Anthony P.; Undem, Bradley J.

    2013-01-01

    A sensory role for ATP was proposed long before general acceptance of its extracellular role. ATP activates and sensitizes signal transmission at multiple sites along the sensory axis, across multiple synapses. P2X and P2Y receptors mediate ATP modulation of sensory pathways and participate in dysregulation, where ATP action directly on primary afferent neurons (PANs), linking receptive field to CNS, has received much attention. Many PANs, especially C-fibers, are activated by ATP, via P2X3-containing trimers. P2X3 knock-out mice and knock-down in rats led to reduced nocifensive activity and visceral reflexes, suggesting that antagonism may offer benefit in sensory disorders. Recently, drug-like P2X3 antagonists, active in a many inflammatory and visceral pain models, have emerged. Significantly, these compounds have no overt CNS action and are inactive versus acute nociception. Selectively targeting ATP sensitization of PANs may lead to therapies that block inappropriate chronic signals at their source, decreasing drivers of peripheral and central wind-up, yet leaving defensive nociceptive and brain functions unperturbed. This article reviews this evidence, focusing on how ATP sensitization of PANs in visceral “hollow” organs primes them to chronic discomfort, irritation and pain (symptoms) as well as exacerbated autonomic reflexes (signs), and how the use of isolated organ-nerve preparations has revealed this mechanism. Urinary and airways systems share many features: dependence on continuous afferent traffic to brainstem centers to coordinate efferent autonomic outflow; loss of descending inhibitory influence in functional and sensory disorders; dependence on ATP in mediating sensory responses to diverse mechanical and chemical stimuli; a mechanistically overlapping array of existing medicines for pathological conditions. These similarities may also play out in terms of future treatment of signs and symptoms, in the potential for benefit of P2X3 antagonists

  11. Cyclophosphamide-Induced Bladder Inflammation Sensitizes and Enhances P2X Receptor Function in Rat Bladder Sensory Neurons

    PubMed Central

    Dang, Khoa; Lamb, Kenneth; Cohen, Michael; Bielefeldt, Klaus; Gebhart, G. F.

    2009-01-01

    We studied sensitization of retrogradely labeled bladder sensory neurons and plasticity of P2X receptor function in a model of cystitis using patch-clamp techniques. Saline (control) or cyclophosphamide (CYP) was given intraperitoneally to rats on days 0, 2, and 4. On day 5, lumbosacral (LS, L6–S2) or thoracolumbar (TL, T12–L2) dorsal root ganglia were removed and dissociated. Bladders from CYP-treated rats showed partial loss of the urothelium and greater myeloperoxidase activity compared with controls. Bladder neurons from CYP-treated rats were increased in size (based on whole cell capacitance) compared with controls and exhibited lower activation threshold, increased action potential width, and greater number of action potentials in response to current injection or application of purinergic agonists. Most control LS bladder neurons (>85%) responded to ATP or α,β-metATP with a slowly desensitizing current; these agonists affected only half of TL neurons, producing predominantly fast/mixed desensitizing currents. CYP treatment increased the fraction of TL bladder neurons sensitive to purinergic agonists (>80%) and significantly increased current density in both LS and TL bladder neurons compared with control. Importantly, LS and TL neurons from CYP-treated rats showed a selective increase in the functional expression of heteromeric P2X2/3 and homomeric P2X3 receptors, respectively. Although desensitizing kinetics were slower in LS neurons from CYP-treated compared with control rats, recovery kinetics were similar. The present results demonstrate that bladder inflammation sensitizes and increases P2X receptor expression and/or function for both pelvic and lumbar splanchnic pathways, which contribute, in part, to the hypersensitivity associated with cystitis. PMID:17959738

  12. Pharmacokinetic and pharmacodynamic profiling of a P2X7 receptor allosteric modulator GSK1482160 in healthy human subjects

    PubMed Central

    Ali, Zahid; Laurijssens, Bart; Ostenfeld, Thor; McHugh, Simon; Stylianou, Anastasia; Scott-Stevens, Paul; Hosking, Louise; Dewit, Odile; Richardson, Jill C; Chen, Chao

    2013-01-01

    AIMS This paper describes findings from the first-in-human study for GSK1482160, an orally available allosteric P2X7 receptor modulator. The study aimed to assess the pharmacokinetics (PK), pharmacodynamics (PD), safety and tolerability of the compound in healthy subjects. METHODS Escalating single doses of up to 1 g were administered to healthy subjects in a single-blind and placebo-controlled fashion. Safety, tolerability, blood drug concentrations and ex vivo Il-1β production in blood were evaluated. RESULTS Drug concentration peaked within 3.5 h of dosing under fasting conditions and declined thereafter with a relatively short half-life of less than 4.5 h. Exposure was proportional to dose with between subject variability of less than 60%. A PK/PD model quantified Il-1β as a function of drug exposure. The model allowed simulation of in vivo pharmacology for various untested dose levels and regimens. Furthermore, the mechanistic model supported the hypothesis that the compound reduces the efficacy of ATP at the P2X7 receptor without affecting its affinity. No major safety or tolerability concerns were identified in this small study (n = 29), except for one case of asymptomatic accelerated idioventricular rhythm at the top dose. CONCLUSION The model-based approach maximized analysis power by integrating all biomarker data and revealed mechanistic insight into the pharmacology of P2X7 modulation by GSK1482160. Simulations by this model ultimately led to the discontinuation of the development of this compound. The therapeutic relevance of the P2X7 receptor remains to be tested in patients. The mechanistic-model-based approach can be applied widely to drug development. PMID:22568863

  13. Identification and characterization of ATP-gated P2X2 receptor gene dominantly expressed in the Japanese flounder (Paralichthys olivaceus) head kidney macrophages.

    PubMed

    Li, Shuo; Chen, Xiaoli; Hao, Gaixiang; Geng, Xuyun; Zhan, Wenbin; Sun, Jinsheng

    2016-07-01

    P2X2 receptor (P2X2R) belongs to the family of purinergic receptors that have been shown to play important roles in regulating host innate immune response. Although the immunologic significance of P2X2R has been studied in mammals, the presence and immune relevance of P2X2R in fish remains unclear. In this study we extended our previous observations by identifying and characterizing a P2X2R ortholog (termed PoP2X2R) from Japanese flounder (Paralichthys olivaceus). Quantitative real-time PCR analysis revealed that PoP2X2R mRNA transcripts are widely distributed in all examined normal tissues and are dominantly expressed in hepatopancreas tissue. In addition, we for the first time showed that multiple P2XR subtypes, including P2X2R, P2X4R and P2X7R are co-expressed in the Japanese flounder head kidney macrophages (HKMs) and peripheral blood lymphocytes (PBLs), indicating that they may assemble into hetero-receptor complex or interact in the form of homotrimers to trigger diverse purinergic signaling in the Japanese flounder immune cells. Compared with the known Japanese flounder P2X4 and P2X7 receptors, however, PoP2X2R is much more abundantly expressed in the Japanese flounder HKM cells, suggesting that PoP2X2R may play an important role in this type of immune cells. Glycosylation and immunohistochemistry analyses revealed that PoP2X2R is a glycoprotein expressed on the plasma membrane. Immune challenges experiments showed that PoP2X2R was significantly induced by LPS, poly(I:C) and zymosan stimulations in the HKM and PBL cells, and by Edwardsiella tarda infections in spleen and gill tissues as well. Taken together, we have identified and characterized a new P2X2R member that is involved in fish innate immune response. PMID:27103003

  14. Pentosan polysulfate preserves renal microvascular P2X1 receptor reactivity and autoregulatory behavior in DOCA-salt hypertensive rats.

    PubMed

    Guan, Zhengrong; Singletary, Sean T; Cha, Haword; Van Beusecum, Justin P; Cook, Anthony K; Pollock, Jennifer S; Pollock, David M; Inscho, Edward W

    2016-03-15

    Inflammation contributes to ANG II-associated impairment of renal autoregulation and microvascular P2X1 receptor signaling, but its role in renal autoregulation in mineralocorticoid-induced hypertension is unknown. Autoregulatory behavior was assessed using the blood-perfused juxtamedullary nephron preparation. Hypertension was induced in uninephrectomized control rats (UNx) by subcutaneous implantation of a DOCA pellet plus administration of 1% NaCl in the drinking water (DOCA-salt) for 3 wk. DOCA-salt rats developed hypertension that was unaltered by anti-inflammatory treatment with pentosan polysulfate (DOCA-salt+PPS) but was suppressed with "triple therapy" (hydrochlorothiazide, hydralazine, and reserpine; DOCA-salt+TTx). Baseline arteriolar diameters were similar across all groups. UNx rats exhibited pressure-dependent vasoconstriction with diameters declining to 69 ± 2% of control at 170 mmHg, indicating intact autoregulation. DOCA-salt treatment significantly blunted this pressure-mediated vasoconstriction. Diameters remained between 91 ± 4 and 98 ± 3% of control over 65-170 mmHg, indicating impaired autoregulation. In contrast, pressure-mediated vasoconstriction was preserved in DOCA-salt+PPS and DOCA-salt+TTx rats, reaching 77 ± 7 and 75 ± 3% of control at 170 mmHg, respectively. ATP is required for autoregulation via P2X1 receptor activation. ATP- and β,γ-methylene ATP (P2X1 receptor agonist)-mediated vasoconstriction were markedly attenuated in DOCA-salt rats compared with UNx (P < 0.05), but significantly improved by PPS or TTx (P < 0.05 vs. DOCA-salt) treatment. Arteriolar responses to adenosine and UTP (P2Y2 receptor agonist) were unaffected by DOCA-salt treatment. PPS and TTx significantly reduced MCP-1 and protein excretion in DOCA-salt rats. These results support the hypothesis that hypertension triggers inflammatory cascades but anti-inflammatory treatment preserves renal autoregulation in DOCA-salt rats, most likely by normalizing renal

  15. Purinergic signaling via P2X7 receptor mediates IL-1β production in Kupffer cells exposed to silica nanoparticle.

    PubMed

    Kojima, Shuji; Negishi, Yusuke; Tsukimoto, Mitsutoshi; Takenouchi, Takato; Kitani, Hiroshi; Takeda, Ken

    2014-07-01

    There is extensive evidence that nanoparticles (NPs) cause adverse effects in multiple organs, including liver, though the mechanisms involved remain to be fully established. Kupffer cells are macrophages resident in the liver, and play important roles in liver inflammation induced by various toxic agents, including lipopolysaccharide (LPS). Interleukin-1 (IL-1) family members IL-1α,β are released from LPS-primed macrophages exposed to NPs, including silica NPs (SNPs), via activation of nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 inflammasomes. Here, we investigated the mechanism of production of IL-1β via activation of inflammasomes in mouse Kupffer cell line KUP5, focusing on the role of purinergic signaling via P2X7 receptor. IL-1β production by LPS-primed KUP5 cells exposed to SNPs was increased dose-dependently, and was greatest in response to SNPs with a diameter of 30 nm (SNP30), as compared with 70-nm and 300-nm SNPs (SNP70 and SNP300). ATP release was also highest in cells exposed to SNP30. Treatment of LPS-primed KUP5 cells with ATP also induced a high level of IL-1β production, similar to that induced by SNP30. IL-1β production was significantly inhibited by apyrase (an ecto-nucleotidase) and A438079 (a P2X7 antagonist/ATP-release inhibitor). Production of reactive oxygen species (ROS) was confirmed in cells exposed to SNP30. In conclusion, ATP released from P2X7 receptor in response to stimulation of KUP5 cells with SNP30 induces ROS production via cell-membrane NADPH oxidase. The ROS causes activation of inflammasomes, leading to caspase-1-dependent processing of IL-1β. PMID:24685903

  16. Upregulated P2X3 Receptor Expression in Patients with Intractable Temporal Lobe Epilepsy and in a Rat Model of Epilepsy.

    PubMed

    Zhou, Xin; Ma, Li-Min; Xiong, Yan; Huang, Hao; Yuan, Jin-Xian; Li, Ruo-Han; Li, Jia-Ni; Chen, Yang-Mei

    2016-06-01

    Purinergic P2X3 receptors (P2X3Rs) play extensive roles in nerve cells in the central nervous system, particularly in hyperexcitability and calcium (Ca(2+)) influx. However, the role of P2X3Rs in epilepsy has not been previously investigated. To determine the relationship between P2X3Rs and epilepsy, the expression and cellular location of P2X3Rs in patients with intractable temporal lobe epilepsy (TLE) and in a lithium chloride-pilocarpine-induced chronic rat model of epilepsy were assessed. Furthermore, the function of P2X3Rs was assessed in vitro. Real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot analysis were used to evaluate the expression levels of P2X3Rs in brain tissues from TLE patients and an epileptic rat model, whereas immunofluorescence labeling was applied to determine the distribution of target proteins. Whole-cell recording was subsequently performed to identify the influence of P2X3Rs on seizure-like discharges. P2X3Rs were located at the cell bodies and dendrites of neurons with significantly increased expression in the TLE patients and epileptic rat model. In vitro, P2X3R activation accelerated sustained repetitive firing, whereas P2X3R inhibition led to relatively low-frequency discharges. To the best of our knowledge, this is the first study provide evidence that upregulated P2X3R expression exists in both epileptic humans and rats and may aggravate the epileptic state in vitro. Thus, P2X3Rs may represent a novel therapeutic target for antiepileptic drugs. PMID:26738991

  17. Pannexin-1 and P2X7-Receptor Are Required for Apoptotic Osteocytes in Fatigued Bone to Trigger RANKL Production in Neighboring Bystander Osteocytes.

    PubMed

    Cheung, Wing Yee; Fritton, J Christopher; Morgan, Stacy Ann; Seref-Ferlengez, Zeynep; Basta-Pljakic, Jelena; Thi, Mia M; Suadicani, Sylvia O; Spray, David C; Majeska, Robert J; Schaffler, Mitchell B

    2016-04-01

    Osteocyte apoptosis is required to induce intracortical bone remodeling after microdamage in animal models, but how apoptotic osteocytes signal neighboring "bystander" cells to initiate the remodeling process is unknown. Apoptosis has been shown to open pannexin-1 (Panx1) channels to release adenosine diphosphate (ATP) as a "find-me" signal for phagocytic cells. To address whether apoptotic osteocytes use this signaling mechanism, we adapted the rat ulnar fatigue-loading model to reproducibly introduce microdamage into mouse cortical bone and measured subsequent changes in osteocyte apoptosis, receptor activator of NF-κB ligand (RANKL) expression and osteoclastic bone resorption in wild-type (WT; C57Bl/6) mice and in mice genetically deficient in Panx1 (Panx1KO). Mouse ulnar loading produced linear microcracks comparable in number and location to the rat model. WT mice showed increased osteocyte apoptosis and RANKL expression at microdamage sites at 3 days after loading and increased intracortical remodeling and endocortical tunneling at day 14. With fatigue, Panx1KO mice exhibited levels of microdamage and osteocyte apoptosis identical to WT mice. However, they did not upregulate RANKL in bystander osteocytes or initiate resorption. Panx1 interacts with P2X7 R in ATP release; thus, we examined P2X7 R-deficient mice and WT mice treated with P2X7 R antagonist Brilliant Blue G (BBG) to test the possible role of ATP as a find-me signal. P2X7 RKO mice failed to upregulate RANKL in osteocytes or induce resorption despite normally elevated osteocyte apoptosis after fatigue loading. Similarly, treatment of fatigued C57Bl/6 mice with BBG mimicked behavior of both Panx1KO and P2X7 RKO mice; BBG had no effect on osteocyte apoptosis in fatigued bone but completely prevented increases in bystander osteocyte RANKL expression and attenuated activation of resorption by more than 50%. These results indicate that activation of Panx1 and P2X7 R are required for apoptotic osteocytes

  18. Inefficient constitutive inhibition of P2X3 receptors by brain natriuretic peptide system contributes to sensitization of trigeminal sensory neurons in a genetic mouse model of familial hemiplegic migraine

    PubMed Central

    Marchenkova, Anna; Vilotti, Sandra; Ntamati, Niels; van den Maagdenberg, Arn MJM

    2016-01-01

    Background On trigeminal ganglion neurons, pain-sensing P2X3 receptors are constitutively inhibited by brain natriuretic peptide via its natriuretic peptide receptor-A. This inhibition is associated with increased P2X3 serine phosphorylation and receptor redistribution to non-lipid raft membrane compartments. The natriuretic peptide receptor-A antagonist anantin reverses these effects. We studied whether P2X3 inhibition is dysfunctional in a genetic familial hemiplegic migraine type-1 model produced by introduction of the human pathogenic R192Q missense mutation into the mouse CACNA1A gene (knock-in phenotype). This model faithfully replicates several properties of familial hemiplegic migraine type-1, with gain-of-function of CaV2.1 Ca2+ channels, raised levels of the algogenic peptide calcitonin gene-related peptide, and enhanced activity of P2X3 receptors in trigeminal ganglia. Results In knock-in neurons, anantin did not affect P2X3 receptor activity, membrane distribution, or serine phosphorylation level, implying ineffective inhibition by the constitutive brain natriuretic peptide/natriuretic peptide receptor-A pathway. However, expression and functional properties of this pathway remained intact together with its ability to downregulate TRPV1 channels. Reversing the familial hemiplegic migraine type-1 phenotype with the CaV2.1-specific antagonist, ω-agatoxin IVA restored P2X3 activity to wild-type level and enabled the potentiating effects of anantin again. After blocking calcitonin gene-related peptide receptors, P2X3 receptors exhibited wild-type properties and were again potentiated by anantin. Conclusions P2X3 receptors on mouse trigeminal ganglion neurons are subjected to contrasting modulation by inhibitory brain natriuretic peptide and facilitatory calcitonin gene-related peptide that both operate via complex intracellular signaling. In the familial hemiplegic migraine type-1 migraine model, the action of calcitonin gene-related peptide appears to

  19. Pharmacological properties of P2X3-receptors present in neurones of the rat dorsal root ganglia

    PubMed Central

    Rae, M G; Rowan, E G; Kennedy, C

    1998-01-01

    The electrophysiological actions of several agonists which may differentiate between P2X1- and P2X3-receptors were studied under concentration and voltage-clamp conditions in dissociated neurones of 1–4 day old rat dorsal root ganglia.β,γ-Methylene-D-ATP (β,γ-me-D-ATP) (1–300 μM), diadenosine 5′,5′′′-P1,P5-pentaphosphate (AP5A) (100 nM–300 μM), diadenosine 5′,5′′′-P1,P4-tetraphosphate (AP4A) (300 nM–300 μM) and uridine 5′-triphosphate (UTP) (1 μM–1 mM) all activated concentration-dependent inward currents with a latency to onset of a few ms.The concentration-response curves for β,γ-me-D-ATP and AP5A and ATP had similar maximum values, while that for AP4A had a lower maximum. The concentration-response curve to UTP was shallow and did not reach a maximum. β,γ-Methylene-L-ATP was virtually inactive. The rank order of agonist potency was ATP>AP5A≈amp;AP4A>β,γ-me-D-ATP>UTP>>β,γ-methylene-L-ATP.The inward currents were inhibited by the P2-receptor antagonists suramin (100 μM) and pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS) (10 μM). PPADS also inhibited responses to ATP (800 nM) and α,β-methylene ATP (2 μM) in a concentration-dependent manner.This study shows that β,γ-me-D-ATP, AP5A, AP4A and UTP all act via a suramin- and PPADS-sensitive P2X-receptor to evoke rapid, transient inward currents in dissociated neurones of rat dorsal root ganglia. The very low activity of β,γ-methylene-L-ATP suggests that the agonists were acting at the P2X3-subtype to produce these effects. PMID:9630357

  20. The purinergic receptor P2X7 role in control of Dengue virus-2 infection and cytokine/chemokine production in infected human monocytes.

    PubMed

    Corrêa, Gladys; de A Lindenberg, Carolina; Fernandes-Santos, Caroline; Gandini, Mariana; Petitinga Paiva, Fabienne; Coutinho-Silva, Robson; F Kubelka, Claire

    2016-07-01

    Purinergic signaling has a crucial role in intracellular pathogen elimination. The P2X7 purinergic receptor (P2X7R), once activated by ATP, leads to pro-inflammatory responses including reactive oxygen species production. ATP can be released by injured cells, as endogenous danger signals. Dengue fever may evolve to a severe disease, leading to hypovolemic shock and coagulation dysfunctions as a result of a cytokine storm. Our aim was to evaluate the role of P2X7R activation during Dengue virus (DENV) infection. Extracellular ATP inhibited viral load in pretreated monocytes, as measured by NS1 secretion and by decrease in DENV(+) P2X7(+) cell frequencies, suggesting that P2X7R is involved in the antiviral response. Nitric oxide (NO) has anti-DENV properties and is decreased after DENV infection. NO production after ATP stimulation is abrogated by KN62 treatment, a specific P2X7R inhibitor, indicating that P2X7R likely is acting in the virus containment process. Additionally, TNF, CXCL8, CCL2 and CXCL10 factors that are associated with dengue severity were modulated by the P2X7R activation. We conclude that P2X7R is directly involved in the modulation of the antiviral and inflammatory process that occurs during DENV infection in vitro, and may have an important role in patient recovery in a first moment. PMID:26969484

  1. LncRNA NONRATT021972 involved the pathophysiologic processes mediated by P2X7 receptors in stellate ganglia after myocardial ischemic injury.

    PubMed

    Zou, Lifang; Tu, Guihua; Xie, Wei; Wen, Shiyao; Xie, Qiuyu; Liu, Shuangmei; Li, Guilin; Gao, Yun; Xu, Hong; Wang, Shouyu; Xue, Yun; Wu, Bing; Lv, Qiulan; Ying, Mofeng; Zhang, Xi; Liang, Shangdong

    2016-03-01

    Adenosine triphosphate (ATP) acts on P2X receptors to initiate signal transmission. P2X7 receptors play a role in the pathophysiological process of myocardial ischemic injury. Long noncoding RNAs (lncRNAs) participate in numerous biological functions independent of protein translation. LncRNAs are implicated in nervous system diseases. This study investigated the effects of NONRATT021972 small interference RNA (siRNA) on the pathophysiologic processes mediated by P2X7 receptors in stellate ganglia (SG) after myocardial ischemic injury. Our results demonstrated that the expression of NONRATT021972 in SG was significantly higher in the myocardial ischemic (MI) group than in the control group. Treatment of MI rats with NONRATT021972 siRNA, the P2X7 antagonist brilliant blue G (BBG), or P2X7 siRNA improved the histology of injured ischemic cardiac tissues and decreased the elevated concentrations of serum myocardial enzymes, creatine kinase (CK), CK isoform MB (CK-MB), lactate dehydrogenase (LDH) and aspartate aminotransferase (AST) compared to the MI rats. NONRATT021972 siRNA, BBG, or P2X7 siRNA treatment in MI rats decreased the expression levels of P2X7 immunoreactivity, P2X7 messenger RNA (mRNA), and P2X7 protein, interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and phosphorylated p38 mitogen-activated protein kinase (p38 MAPK) in the SG compared to MI rats. NONRATT021972 siRNA treatment prevented the pathophysiologic processes mediated by P2X7 receptors in the SG after myocardial ischemic injury. PMID:26630943

  2. Vatalanib decrease the positive interaction of VEGF receptor-2 and P2X2/3 receptor in chronic constriction injury rats.

    PubMed

    Liu, Shuangmei; Xu, Changshui; Li, Guilin; Liu, Han; Xie, Jinyan; Tu, Guihua; Peng, Haiying; Qiu, Shuyi; Liang, Shangdong

    2012-05-01

    Neuropathic pain can arise from a lesion affecting the peripheral nervous system. Selective P2X(3) and P2X(2/3) receptors' antagonists effectively reduce neuropathic pain. VEGF inhibitors are effective for pain relief. The present study investigated the effects of Vatalanib (VEGF receptor-2 (VEGFR-2) inhibitor) on the neuropathic pain to address the interaction of VEGFR-2 and P2X(2/3) receptor in dorsal root ganglia of chronic constriction injury (CCI) rats. Neuropathic pain symptoms following CCI are similar to most peripheral lesions as assessed by the Neuropathic Pain Symptom Inventory. Sprague-Dawley rats were randomly divided into sham group, CCI group and CCI rats treated with Vatalanib group. Mechanical withdrawal threshold and thermal withdrawal latency were measured. Co-expression of VEGFR-2 and P2X(2) or P2X(3) in L4-6 dorsal root ganglia (DRG) was detected by double-label immunofluorescence. The modulation effect of VEGF on P2X(2/3) receptor agonist-activated currents in freshly isolated DRG neurons of rats both of sham and CCI rats was recorded by whole-cell patch-clamp technique. The mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) in CCI group were lower than those in sham group (p<0.05). MWT and TWL in CCI rats treated with Vatalanib group were increased compared with those in CCI group (p<0.05). VEGFR-2 and P2X(2) or P2X(3) receptors were co-expressed in the cytoplasm and surface membranes of DRG. The co-expression of VEGFR-2 and P2X(2) or P2X(3) receptor in CCI group exhibited more intense staining than those in sham group and CCI rats treated with Vatalanib group, respectively. VEGF enhanced the amplitude of ATP and α,β-meATP -activated currents of both sham and CCI rats. Increment effects of VEGF on ATP and α,β-meATP -activated currents in CCI rats were higher than those in sham rats. Both ATP (100 μM) and α,β-meATP (10 μM)- activated currents enhanced by VEGF ( 1nM) were significantly blocked by Vatalanib (1

  3. Structure Activity Relationship of (N)-Methanocarba Phosphonate Analogues of 5’-AMP as Cardioprotective Agents Acting Through a Cardiac P2X Receptor

    PubMed Central

    Kumar, T. Santhosh; Zhou, Si-Yuan; Joshi, Bhalchandra V.; Balasubramanian, Ramachandran; Yang, Tiehong; Liang, Bruce T.; Jacobson, Kenneth A.

    2010-01-01

    P2X receptor activation protects in heart failure models. MRS2339 3, a 2-chloro-AMP derivative containing a (N)-methanocarba (bicyclo[3.1.0]hexane) system, activates this cardioprotective channel. Michaelis–Arbuzov and Wittig reactions provided phosphonate analogues of 3, expected to be stable in vivo due to the C-P bond. After chronic administration via a mini-osmotic pump (Alzet), some analogues significantly increased intact heart contractile function in calsequestrin-overexpressing mice (genetic model of heart failure) compared to vehicle-infused mice (all inactive at the vasodilatory P2Y1 receptor). Two phosphonates, (1’S,2’R,3’S,4’R,5’S)-4’-(6-amino-2-chloropurin-9-yl)-2’,3’-(dihydroxy)-1’-(phosphonomethylene)-bicyclo[3.1.0]hexane 4 and its homologue 9, both 5’-saturated, containing a 2-Cl substitution, improved echocardiography-derived fractional shortening (20.25% and 19.26%, respectively, versus 13.78% in controls), while unsaturated 5’-extended phosphonates, all 2-H analogues, and a CH3-phosphonate were inactive. Thus, chronic administration of nucleotidase-resistant phosphonates conferred a beneficial effect, likely via cardiac P2X receptor activation. Thus, we have greatly expanded the range of carbocyclic nucleotide analogues that represent potential candidates for the treatment of heart failure. PMID:20192270

  4. The role of P2X7 purinergic receptors in inflammatory and nociceptive changes accompanying cyclophosphamide-induced haemorrhagic cystitis in mice

    PubMed Central

    Martins, JP; Silva, RBM; Coutinho-Silva, R; Takiya, CM; Battastini, AMO; Morrone, FB; Campos, MM

    2012-01-01

    BACKGROUND AND PURPOSE ATP is released in response to cellular damage, and P2X7 receptors have an essential role in the onset and maintenance of pathological changes. Haemorrhagic cystitis (HC) is a well-known adverse effect of therapy with cyclophosphamide used for the treatment of many solid tumours and autoimmune conditions. Here we have evaluated the role of P2X7 receptors in a model of HC induced by cyclophosphamide. EXPERIMENTAL APPROACH Effects of pharmacological antagonism or genetic deletion of P2X7 receptor on cyclophosphamide-induced HC in mice was assessed by nociceptive and inflammatory measures. In addition, the presence of immunoreactive P2X7 receptors was assessed by immunohistochemistry. KEY RESULTS Pretreatment with the selective P2X7 receptor antagonist A-438079 or genetic ablation of P2X7 receptors reduced nociceptive behaviour scores in the HC model. The same strategies decreased both oedema and haemorrhage indices, on macroscopic or histological evaluation. Treatment with A-438079 decreased the staining for c-Fos in the lumbar spinal cord and brain cortical areas. Treatment with A-438079 also prevented the increase of urinary bladder myeloperoxidase activity and macrophage migration induced by cyclophosphamide and reduced the tissue levels of IL-1β and TNF-α. Finally, P2X7 receptors were markedly up-regulated in the bladders of mice with cyclophosphamide-induced HC. CONCLUSIONS AND IMPLICATIONS P2X7 receptors were significantly involved in a model of HC induced by cyclophosphamide. Pharmacological inhibition of these receptors might represent a new therapeutic option for this pathological condition. PMID:21675966

  5. Expression of the ATP-gated P2X7 Receptor on M Cells and Its Modulating Role in the Mucosal Immune Environment.

    PubMed

    Kim, Sae-Hae; Lee, Ha-Yan; Jang, Yong-Suk

    2015-02-01

    Interactions between microbes and epithelial cells in the gastrointestinal tract are closely associated with regulation of intestinal mucosal immune responses. Recent studies have highlighted the modulation of mucosal immunity by microbe-derived molecules such as ATP and short-chain fatty acids. In this study, we undertook to characterize the expression of the ATP-gated P2X7 receptor (P2X7R) on M cells and its role in gastrointestinal mucosal immune regulation because it was poorly characterized in Peyer's patches, although purinergic signaling via P2X7R and luminal ATP have been considered to play an important role in the gastrointestinal tract. Here, we present the first report on the expression of P2X7R on M cells and characterize the role of P2X7R in immune enhancement by ATP or LL-37. PMID:25713508

  6. Insights into the Molecular Mechanisms Underlying Mammalian P2X7 Receptor Functions and Contributions in Diseases, Revealed by Structural Modeling and Single Nucleotide Polymorphisms

    PubMed Central

    Jiang, Lin-Hua; Baldwin, Jocelyn M.; Roger, Sebastien; Baldwin, Stephen A.

    2013-01-01

    The mammalian P2X7 receptors (P2X7Rs), a member of the ionotropic P2X receptor family with distinctive functional properties, play an important part in mediating extracellular ATP signaling in health and disease. A clear delineation of the molecular mechanisms underlying the key receptor properties, such as ATP-binding, ion permeation, and large pore formation of the mammalian P2X7Rs, is still lacking, but such knowledge is crucial for a better understanding of their physiological functions and contributions in diseases and for development of therapeutics. The recent breakthroughs in determining the atomic structures of the zebrafish P2X4.1R in the closed and ATP-bound open states have provided the long-awaited structural information. The human P2RX7 gene is abundant with non-synonymous single nucleotide polymorphisms (NS-SNPs), which generate a repertoire of human P2X7Rs with point mutations. Characterizations of the NS-SNPs identified in patients of various disease conditions and the resulting mutations have informed previously unknown molecular mechanisms determining the mammalian P2X7R functions and diseases. In this review, we will discuss the new insights into such mechanisms provided by structural modeling and recent functional and genetic linkage studies of NS-SNPs. PMID:23675347

  7. P2X2 Receptor Terminal Field Demarcates a "Transition Zone" for Gustatory and Mechanosensory Processing in the Mouse Nucleus Tractus Solitarius.

    PubMed

    Breza, Joseph M; Travers, Susan P

    2016-07-01

    Peripheral gustatory neurons express P2X2 purinergic receptors and terminate in the rostral portion of the nucleus tractus solitarius (rNTS), but a relationship between the P2X2 terminal field and taste evoked activity has not been established. Additionally, a portion of somatosensory neurons from the trigeminal nerve, which are devoid of P2X2 expression, also terminate in the lateral rNTS. We hypothesized that P2X2 receptor expression on afferent nerve endings could be used as an anatomical tool for segregating gustatory from mechanosensory responsive regions in the mouse rNTS. C57BL/6 mice were used to record extracellular activity from neurons within the rNTS and the laterally adjacent reticular formation and trigeminal nucleus. Histological reconstruction of electrolytic lesions indicated that gustatory activity coincided with electrode tracks that traversed through P2X2 terminal fields. Gustatory recordings made more rostral in the rNTS had receptive fields located in the anterior oral cavity (AO), whereas gustatory recordings made more caudal in the rNTS had receptive fields located in the posterior oral cavity (PO). Mechanosensory neurons with AO receptive fields were recorded near the lateral border of the P2X2 terminal field and became numerous on electrode tracks made lateral to the P2X2 terminal field. In contrast, mechanosensory responses with PO receptive fields were recorded within the P2X2 terminal field along with gustatory activity and transitioned to mechanosensory only outside the P2X2 terminal field. Collectively, our results indicate that the lateral border of the P2X2 terminal field, demarcates a faithful "transition zone," where AO responses transition from gustatory to mechanosensory. PMID:27131102

  8. P2X7 Receptor Suppression Preserves Blood-Brain Barrier through Inhibiting RhoA Activation after Experimental Intracerebral Hemorrhage in Rats.

    PubMed

    Zhao, Hengli; Zhang, Xuan; Dai, Zhiqiang; Feng, Yang; Li, Qiang; Zhang, John H; Liu, Xin; Chen, Yujie; Feng, Hua

    2016-01-01

    Blockading P2X7 receptor(P2X7R) provides neuroprotection toward various neurological disorders, including stroke, traumatic brain injury, and subarachnoid hemorrhage. However, whether and how P2X7 receptor suppression protects blood-brain barrier(BBB) after intracerebral hemorrhage(ICH) remains unexplored. In present study, intrastriatal autologous-blood injection was used to mimic ICH in rats. Selective P2X7R inhibitor A438079, P2X7R agonist BzATP, and P2X7R siRNA were administrated to evaluate the effects of P2X7R suppression. Selective RhoA inhibitor C3 transferase was administered to clarify the involvement of RhoA. Post-assessments, including neurological deficits, Fluoro-Jade C staining, brain edema, Evans blue extravasation and fluorescence, western blot, RhoA activity assay and immunohistochemistry were performed. Then the key results were verified in collagenase induced ICH model. We found that endogenous P2X7R increased at 3 hrs after ICH with peak at 24 hrs, then returned to normal at 72 hrs after ICH. Enhanced immunoreactivity was observed on the neurovascular structure around hematoma at 24 hrs after ICH, along with perivascular astrocytes and endothelial cells. Both A438079 and P2X7R siRNA alleviated neurological deficits, brain edema, and BBB disruption after ICH, in association with RhoA activation and down-regulated endothelial junction proteins. However, BzATP abolished those effects. In addition, C3 transferase reduced brain injury and increased endothelial junction proteins' expression after ICH. These data indicated P2X7R suppression could preserve BBB integrity after ICH through inhibiting RhoA activation. PMID:26980524

  9. P2X7 Receptor Suppression Preserves Blood-Brain Barrier through Inhibiting RhoA Activation after Experimental Intracerebral Hemorrhage in Rats

    PubMed Central

    Zhao, Hengli; Zhang, Xuan; Dai, Zhiqiang; Feng, Yang; Li, Qiang; Zhang, John H.; Liu, Xin; Chen, Yujie; Feng, Hua

    2016-01-01

    Blockading P2X7 receptor(P2X7R) provides neuroprotection toward various neurological disorders, including stroke, traumatic brain injury, and subarachnoid hemorrhage. However, whether and how P2X7 receptor suppression protects blood-brain barrier(BBB) after intracerebral hemorrhage(ICH) remains unexplored. In present study, intrastriatal autologous-blood injection was used to mimic ICH in rats. Selective P2X7R inhibitor A438079, P2X7R agonist BzATP, and P2X7R siRNA were administrated to evaluate the effects of P2X7R suppression. Selective RhoA inhibitor C3 transferase was administered to clarify the involvement of RhoA. Post-assessments, including neurological deficits, Fluoro-Jade C staining, brain edema, Evans blue extravasation and fluorescence, western blot, RhoA activity assay and immunohistochemistry were performed. Then the key results were verified in collagenase induced ICH model. We found that endogenous P2X7R increased at 3 hrs after ICH with peak at 24 hrs, then returned to normal at 72 hrs after ICH. Enhanced immunoreactivity was observed on the neurovascular structure around hematoma at 24 hrs after ICH, along with perivascular astrocytes and endothelial cells. Both A438079 and P2X7R siRNA alleviated neurological deficits, brain edema, and BBB disruption after ICH, in association with RhoA activation and down-regulated endothelial junction proteins. However, BzATP abolished those effects. In addition, C3 transferase reduced brain injury and increased endothelial junction proteins’ expression after ICH. These data indicated P2X7R suppression could preserve BBB integrity after ICH through inhibiting RhoA activation. PMID:26980524

  10. Primary and secondary agonists can use P2X1 receptors as a major pathway to increase intracellular Ca2+ in the human platelet

    PubMed Central

    FUNG, C Y E; CENDANA, C; FARNDALE, R W; MAHAUT-SMITH, M P

    2007-01-01

    See also Nurden AT. Does ATP act through P2X1 receptors to regulate platelet activation and thrombus formation? This issue, pp 907–9. In the platelet, it is well established that many G-protein- and tyrosine kinase-coupled receptors stimulate phospholipase-C-dependent Ca2+ mobilization; however, the extent to which secondary activation of adenosine 5′-triphosphate (ATP)-gated P2X1 receptors contributes to intracellular Ca2+ responses remains unclear. We now show that selective inhibition of P2X1 receptors substantially reduces the [Ca2+]i increase evoked by several important agonists in human platelets; for collagen, thromboxane A2, thrombin, and adenosine 5′-diphoshate (ADP) the maximal effect was a reduction to 18%, 34%, 52%, and 69% of control, respectively. The direct contribution of P2X1 to the secondary Ca2+ response was far greater than that of either P2Y receptors activated by co-released ADP, or via synergistic P2X1:P2Y interactions. The relative contribution of P2X1 to the peak Ca2+ increase varied with the strength of the initial stimulus, being greater at low compared to high levels of stimulation for both glycoprotein VI and PAR-1, whereas P2X1 contributed equally at both low and high levels of stimulation of thromboxane A2 receptors. In contrast, only strong stimulation of P2Y receptors resulted in significant P2X1 receptor activation. ATP release was detected by soluble luciferin:luciferase in response to all agonists that stimulated secondary P2X1 receptor activation. However, P2X1 receptors were stimulated earlier and to a greater extent than predicted from the average ATP release, which can be accounted for by a predominantly autocrine mechanism of activation. Given the central role of [Ca2+]i increases in platelet activation, these studies indicate that ATP should be considered alongside ADP and thromboxane A2 as a significant secondary platelet agonist. PMID:17362227

  11. F-actin links Epac-PKC signaling to purinergic P2X3 receptor sensitization in dorsal root ganglia following inflammation

    PubMed Central

    Gu, Yanping; Wang, Congying; Li, GuangWen

    2016-01-01

    Sensitization of purinergic P2X3 receptors (P2X3Rs) contributes to the production of exaggerated nociceptive responses following inflammatory injury. We showed previously that prostaglandin E2 (PGE2) potentiates P2X3R-mediated ATP currents in dorsal root ganglion neurons isolated from both control and complete Freund’s adjuvant-induced inflamed rats. PGE2 potentiation of ATP currents depends only on PKA signaling in control neurons, but it depends on both PKA and PKC signaling in inflamed neurons. We further found that inflammation evokes an increase in exchange proteins directly activated by cAMP (Epacs) in dorsal root ganglions. This increase promotes the activation of PKC to produce a much enhanced PGE2 effect on ATP currents and to elicit Epac-dependent flinch nocifensive behavioral responses in complete Freund’s adjuvant rats. The link between Epac-PKC signaling and P2X3R sensitization remains unexplored. Here, we show that the activation of Epacs promotes the expression of phosphorylated PKC and leads to an increase in the cytoskeleton, F-actin, expression at the cell perimeter. Depolymerization of F-actin blocks PGE2-enhanced ATP currents and inhibits P2X3R-mediated nocifensive responses after inflammation. Thus, F-actin is dynamically involved in the Epac-PKC-dependent P2X3R sensitization. Furthermore, Epacs induce a PKC-dependent increase in the membrane expression of P2X3Rs. This increase is abolished by F-actin depolymerization, suggesting that F-actin mediates Epac-PKC signaling of P2X3R membrane expression. Thus, after inflammation, an Epac-PKC dependent increase in F-actin in dorsal root ganglion neurons enhances the membrane expression of P2X3Rs to bring about sensitization of P2X3Rs and abnormal pain behaviors. PMID:27385722

  12. Rho/ROCK acts downstream of lysophosphatidic acid receptor 1 in modulating P2X3 receptor-mediated bone cancer pain in rats

    PubMed Central

    Wu, Jing-xiang; Yuan, Xiao-min; Wang, Qiong; Wei, Wang

    2016-01-01

    Background Lysophosphatidic acid receptor 1 and Rho/ROCK signaling is implicated in bone cancer pain development. However, it remains unknown whether the two signaling pathways function together in P2X3 receptor-mediated bone cancer pain. Results In this study, using a rat model of bone cancer, we examined the expression of P2X3 and lysophosphatidic acid receptor 1 in rat dorsal root ganglion neurons and further dissected whether lysophosphatidic acid receptor 1 and Rho/ROCK-mediated pathways interacted in modulating rat pain behavior. Bone cancer was established by inoculating Walker 256 cells into the left tibia of female Wistar rats. We observed a gradual and yet significant decline in mean paw withdrawal threshold in rats with bone cancer, but not in control rats. Our immunohistochemical staining revealed that the number of P2X3- and lysophosphatidic acid receptor 1-positive dorsal root ganglion neurons was significantly greater in rats with bone cancer than control rats. Lysophosphatidic acid receptor 1 blockade with VPC32183 significantly attenuated decline in mean paw withdrawal threshold. Flinching behavior test further showed that lysophosphatidic acid receptor 1 inhibition with VPC32183 transiently but significantly attenuated α,β-meATP-induced increase in paw lift time per minute. Rho inhibition by intrathecal BoTXC3 caused a rapid reversal in decline in mean paw withdrawal threshold of rats with bone cancer. Flinching behavior test showed that BoTXC3 transiently and significantly attenuated α,β-meATP-induced increase in paw lift time per minute. Similar findings were observed with ROCK inhibition by intrathecal Y27632. Furthermore, VPC32183 and BoTXC3 effectively aborted the appearance of lysophosphatidic acid-induced calcium influx peak. Conclusions Lysophosphatidic acid and its receptor LPAR1, acting through the Rho-ROCK pathway, regulate P2X3 receptor in the development of both mechanical and spontaneous pain in bone cancer. PMID:27094551

  13. P2X1 receptor inhibition and soluble CD39 administration as novel approaches to widen the cardiovascular therapeutic window

    PubMed Central

    Fung, C.Y. Eleanor; Marcus, Aaron J.; Broekman, M. Johan; Mahaut-Smith, Martyn P.

    2010-01-01

    Thrombus formation at sites of disrupted atherosclerotic plaques is a leading cause of death and disability worldwide. Although the platelet is now recognized to be a central regulator of thrombus formation, development of antiplatelet reagents that selectively target thrombosis over hemostasis represents a challenge. Existing prophylactic antiplatelet therapies are centered on the use of aspirin, an irreversible cyclo-oxygenase inhibitor, and a thienopyridine such as clopidogrel (Plavix), which inactivates the ADP-stimulated P2Y12 receptor. Whilst these compounds are widely used and have beneficial effects for patients, their antithrombotic benefit is complicated by an elevated bleeding risk and substantial or partial “resistance”. Moreover, combination therapy with these two drugs increases the hemorrhagic risk even further. This review explores the possibility of inhibiting the platelet-surface ionotropic P2X1 receptor and/or elevating CD39/NTPDase1 activity as new therapeutic approaches to reduce overall platelet reactivity and recruitment of surrounding platelets at pro-thrombotic locations. Since both proteins affect platelet activation at an early stage in the events leading to thrombosis, but are less crucial in hemostasis, they provide new strategies to widen the cardiovascular therapeutic window without compromising safety. PMID:19467446

  14. Characterization of Ca2+ influx through recombinant P2X receptor in C6BU-1 cells

    PubMed Central

    Ueno, Shinya; Koizumi, Shuichi; Inoue, Kazuhide

    1998-01-01

    The effects of exogenous adenosine 5′-triphosphate (ATP) and α,β-methylene ATP (α,βmeATP) on C6BU-1 cells transfected with P2X2 and P2X3 subtypes, separately or together (P2X2+3), were investigated using fura-2 fluorescence recording and whole-cell patch clamp recording methods.Untransfected C6BU-1 cells showed no intracellular Ca2+ ([Ca2+]i) increase in response to depolarizing stimulation with high K+ or stimulation with ATP. There was no current induced by ATP under voltage clamp conditions in untransfected C6BU-1 cells. ATP caused Ca2+ influx only from extracellular sources in C6BU-1 cells transfected with the P2X subtypes, suggesting that the C6BU-1 cell line is suitable for the characterization of Ca2+ influx through the P2X subtypes.In C6BU-1 cells transfected with the P2X2 subtype, ATP (more than 10 μM) but not α,βmeATP (up to 100 μM) evoked a rise in [Ca2+]i.In the cells transfected with the P2X3 subtype, current responses under voltage clamp conditions were observed at ATP concentrations higher than 0.1 μM of α,βmeATP were required. This discrepancy in the concentration dependence of the agonist responses with respect to the [Ca2+]i rise and the current response was seen only with the P2X3 subtype. In addition, the agonist-induced rise in [Ca2+]i was observed only after the first application because of desensitization of this subtype.In C6BU-1 cells co-transfected with P2X2 and P2X3, ATP at 1 μM evoked a [Ca2+]i rise. This responsiveness was higher than that of the other subtype combinations tested. The efficiency of expression was improved by co-transfection with P2X2 and P2X3, when compared to transfection with the P2X3 subtype alone. The desensitization of the P2X2+3 was apparently slower than that of the P2X3 subtype alone. Therefore, this combination could respond to the repeated application of agonists each time with a [Ca2+]i rise.These results suggest that the P2X2 and P2X3 subtypes assemble a heteromultimer and that this

  15. The azetidine derivative, KHG26792 protects against ATP-induced activation of NFAT and MAPK pathways through P2X7 receptor in microglia.

    PubMed

    Kim, Eun-A; Cho, Chang Hun; Kim, Jiae; Hahn, Hoh-Gyu; Choi, Soo Young; Yang, Seung-Ju; Cho, Sung-Woo

    2015-12-01

    Azetidine derivatives are of interest for drug development because they may be useful therapeutic agents. However, their mechanisms of action remain to be completely elucidated. Here, we have investigated the effects of 3-(naphthalen-2-yl(propoxy)methyl)azetidine hydrochloride (KHG26792) on ATP-induced activation of NFAT and MAPK through P2X7 receptor in the BV-2 mouse microglial cell line. KHG26792 decreased ATP-induced TNF-α release from BV-2 microglia by suppressing, at least partly, P2X7 receptor stimulation. KHG26792 also inhibited the ATP-induced increase in IL-6, PGE2, NO, ROS, CXCL2, and CCL3. ATP induced NFAT activation through P2X7 receptor, with KHG26792 reducing the ATP-induced NFAT activation. KHG26792 inhibited an ATP-induced increase in iNOS protein and ERK phosphorylation. KHG26792 prevented an ATP-induced increase in MMP-9 activity through the P2X7 receptor as a result of degradation of TIMP-1 by cathepsin B. Our data provide mechanistic insights into the role of KHG26792 in the inhibition of TNF-α produced via P2X7 receptor-mediated activation of NFAT and MAPK pathways in ATP-treated BV-2 cells. This study highlights the potential use of KHG26792 as a therapeutic agent for the many diseases of the CNS related to activated microglia. PMID:26522449

  16. Heteromeric assembly of P2X subunits

    PubMed Central

    Saul, Anika; Hausmann, Ralf; Kless, Achim; Nicke, Annette

    2013-01-01

    Transcripts and/or proteins of P2X receptor (P2XR) subunits have been found in virtually all mammalian tissues. Generally more than one of the seven known P2X subunits have been identified in a given cell type. Six of the seven cloned P2X subunits can efficiently form functional homotrimeric ion channels in recombinant expression systems. This is in contrast to other ligand-gated ion channel families, such as the Cys-loop or glutamate receptors, where homomeric assemblies seem to represent the exception rather than the rule. P2XR mediated responses recorded from native tissues rarely match exactly the biophysical and pharmacological properties of heterologously expressed homomeric P2XRs. Heterotrimerization of P2X subunits is likely to account for this observed diversity. While the existence of heterotrimeric P2X2/3Rs and their role in physiological processes is well established, the composition of most other P2XR heteromers and/or the interplay between distinct trimeric receptor complexes in native tissues is not clear. After a description of P2XR assembly and the structure of the intersubunit ATP-binding site, this review summarizes the distribution of P2XR subunits in selected mammalian cell types and the biochemically and/or functionally characterized heteromeric P2XRs that have been observed upon heterologous co-expression of P2XR subunits. We further provide examples where the postulated heteromeric P2XRs have been suggested to occur in native tissues and an overview of the currently available pharmacological tools that have been used to discriminate between homo- and heteromeric P2XRs. PMID:24391538

  17. Activation of P2X(7) receptors stimulates the expression of P2Y(2) receptor mRNA in astrocytes cultured from rat brain.

    PubMed

    D'Alimonte, I; Ciccarelli, R; Di Iorio, P; Nargi, E; Buccella, S; Giuliani, P; Rathbone, M P; Jiang, S; Caciagli, F; Ballerini, P

    2007-01-01

    Under pathological conditions brain cells release ATP at concentrations reported to activate P2X(7) ionotropic receptor subtypes expressed in both neuronal and glial cells. In the present study we report that the most potent P2X(7) receptor agonist BzATP stimulates the expression of the metabotropic ATP receptor P2Y(2) in cultured rat brain astrocytes. In other cell types several kinds of stimulation, including stress or injury, induce P2Y(2) expression that, in turn, is involved in different cell reactions. Similarly, it has recently been found that in astrocytes and astrocytoma cells P2Y(2) sites can trigger neuroprotective pathways through the activation of several mechanisms, including the induction of genes for antiapoptotic factors, neurotrophins, growth factors and neuropeptides. Here we present evidence that P2Y(2) mRNA expression in cultured astrocytes peaks 6 h after BzATP exposure and returns to basal levels after 24 h. This effect was mimicked by high ATP concentrations (1 mM) and was abolished by P2X(7)-antagonists oATP and BBG. The BzATP-evoked P2Y(2) receptor up-regulation in cultured astrocytes was coupled to an increased UTP-mediated intracellular calcium response. This effect was inhibited by oATP and BBG and by P2Y(2)siRNA, thus supporting evidence of increased P2Y(2) activity. To further investigate the mechanisms by which P2X(7) receptors mediated the P2Y(2) mRNA up-regulation, the cells were pre-treated with the chelating agent EGTA, or with inhibitors of mitogen-activated kinase (MAPK) (PD98059) or protein kinase C, (GF109203X). Each inhibitor significantly reduced the extent to which BzATP induced P2Y(2) mRNA. Both BzATP and ATP (1 mM) increased ERK1/2 activation. P2X(7)-induced ERK1/2 phosphorylation was unaffected by pre-treatment of astrocytes with EGTA whereas it was inhibited by GF109203X. Phorbol-12-myristate-13-acetate (PMA), an activator of PKCs, rapidly increased ERK1/2 activation. We conclude that activation of P2X(7) receptors in

  18. Sensitization of P2X3 receptors by cystathionine β-synthetase mediates persistent pain hypersensitivity in a rat model of lumbar disc herniation.

    PubMed

    Wang, Qianliang; Zhu, Hongyan; Zou, Kang; Yuan, Bo; Zhou, You-Lang; Jiang, Xinghong; Yan, Jun; Xu, Guang-Yin

    2015-01-01

    Lumbar disc herniation (LDH) is a major cause of discogenic low back pain and sciatica, but the underlying mechanisms remain largely unknown. Hydrogen sulfide (H2S) is becoming recognized for its involvement in a wide variety of processes including inflammation and nociception. The present study was designed to investigate the roles of the H2S signaling pathway in the regulation of expression and function of purinergic receptors (P2XRs) in dorsal root ganglion (DRG) neurons from rats with LDH. LDH was induced by implantation of autologous nucleus pulposus (NP), harvested from rat tail, in lumbar 5 and 6 spinal nerve roots. Implantation of autologous NP induced persistent pain hypersensitivity, which was partially reversed by an intrathecal injection of A317491, a potent inhibitor of P2X3Rs and P2X2/3Rs. The NP induced persistent pain hypersensitivity was associated with the increased expression of P2X3Rs, but not P2X1Rs and P2X2Rs, receptors in L5-6 DRGs. NP implantation also produced a 2-fold increase in ATP-induced intracellular calcium signals in DRG neurons when compared to those of controls (P < 0.05). Interestingly, NP implantation significantly enhanced expression of the endogenous hydrogen sulfide producing enzyme, cystathionine-β-synthetase (CBS). Systematic administration of O-(Carboxymethyl) hydroxylamine hemihydrochloride (AOAA), an inhibitor of CBS, suppressed the upregulation of P2X3R expression and the potentiation of ATP-induced intracellular calcium signals in DRG neurons (P < 0.05). Intrathecal injection of AOAA markedly attenuated NP induced- persistent pain hypersensitivity. Our results suggest that sensitization of P2X3Rs, which is likely mediated by CBS-H2S signaling in primary sensory neurons, contributes to discogenic pain. Targeting CBS/H2S-P2X3R signaling may represent a potential treatment for neuropathic pain caused by LDH. PMID:25885215

  19. P2X7 receptor blockade protects against cisplatin-induced nephrotoxicity in mice by decreasing the activities of inflammasome components, oxidative stress and caspase-3

    SciTech Connect

    Zhang, Yuanyuan; Yuan, Fahuan; Cao, Xuejiao; Zhai, Zhifang; Gang Huang; Du, Xiang; Wang, Yiqin; Zhang, Jingbo; Huang, Yunjian; Zhao, Jinghong; Hou, Weiping

    2014-11-15

    Nephrotoxicity is a common complication of cisplatin chemotherapy and thus limits the use of cisplatin in clinic. The purinergic 2X7 receptor (P2X7R) plays important roles in inflammation and apoptosis in some inflammatory diseases; however, its roles in cisplatin-induced nephrotoxicity remain unclear. In this study, we first assessed the expression of P2X7R in cisplatin-induced nephrotoxicity in C57BL/6 mice, and then we investigated the changes of renal function, histological injury, inflammatory response, and apoptosis in renal tissues after P2X7R blockade in vivo using an antagonist A-438079. Moreover, we measured the changes of nod-like receptor family, pyrin domain containing proteins (NLRP3) inflammasome components, oxidative stress, and proapoptotic genes in renal tissues in cisplatin-induced nephrotoxicity after treatment with A-438079. We found that the expression of P2X7R was significantly upregulated in the renal tubular epithelial cells in cisplatin-induced nephrotoxicity compared with that of the normal control group. Furthermore, pretreatment with A-438079 markedly attenuated the cisplatin-induced renal injury while lightening the histological damage, inflammatory response and apoptosis in renal tissue, and improved the renal function. These effects were associated with the significantly reduced levels of NLRP3 inflammasome components, oxidative stress, p53 and caspase-3 in renal tissues in cisplatin-induced nephrotoxicity. In conclusions, our studies suggest that the upregulated activity of P2X7R might play important roles in the development of cisplatin-induced nephrotoxicity, and P2X7R blockade might become an effective therapeutic strategy for this disease. - Highlights: • The P2X7R expression was markedly upregulated in cisplatin-induced nephrotoxicity. • P2X7R blockade significantly attenuated the cisplatin-induced renal injury. • P2X7R blockade reduced activities of NLRP3 inflammasome components in renal tissue. • P2X7R blockade

  20. P2X7 receptor-NADPH oxidase axis mediates protein radical formation and Kupffer cell activation in carbon tetrachloride-mediated steatohepatitis in obese mice.

    PubMed

    Chatterjee, Saurabh; Rana, Ritu; Corbett, Jean; Kadiiska, Maria B; Goldstein, Joyce; Mason, Ronald P

    2012-05-01

    While some studies show that carbon tetrachloride-mediated metabolic oxidative stress exacerbates steatohepatitic-like lesions in obese mice, the redox mechanisms that trigger the innate immune system and accentuate the inflammatory cascade remain unclear. Here we have explored the role of the purinergic receptor P2X7-NADPH oxidase axis as a primary event in recognizing the heightened release of extracellular ATP from CCl(4)-treated hepatocytes and generating redox-mediated Kupffer cell activation in obese mice. We found that an underlying condition of obesity led to the formation of protein radicals and posttranslational nitration, primarily in Kupffer cells, at 24h post-CCl(4) administration. The free radical-mediated oxidation of cellular macromolecules, which was NADPH oxidase and P2X7 receptor-dependent, correlated well with the release of TNF-α and MCP-2 from Kupffer cells. The Kupffer cells in CCl(4)-treated mice exhibited increased expression of MHC Class II proteins and showed an activated phenotype. Increased expression of MHC Class II was inhibited by the NADPH oxidase inhibitor apocynin , P2X7 receptor antagonist A438709 hydrochloride, and genetic deletions of the NADPH oxidase p47 phox subunit or the P2X7 receptor. The P2X7 receptor acted upstream of NADPH oxidase activation by up-regulating the expression of the p47 phox subunit and p47 phox binding to the membrane subunit, gp91 phox. We conclude that the P2X7 receptor is a primary mediator of oxidative stress-induced exacerbation of inflammatory liver injury in obese mice via NADPH oxidase-dependent mechanisms. PMID:22343416

  1. Transcriptional Control Mechanisms Associated with the Nucleotide Receptor P2X7, a Critical Regulator of Immunologic, Osteogenic and Neurologic Functions

    PubMed Central

    Lenertz, Lisa Y.; Gavala, Monica L.; Zhu, Yiming; Bertics, Paul J.

    2011-01-01

    The nucleotide receptor P2X7 is an attractive therapeutic target and potential biomarker for multiple inflammatory and neurologic disorders, and it is expressed in several immune, osteogenic and neurologic cell types. Aside from its role in the nervous system, it is activated by ATP released at sites of tissue damage, inflammation and infection. Ligand binding to P2X7 stimulates many cell responses, including calcium fluxes, MAPK activation, inflammatory mediator release, and apoptosis. Much work has centered on P2X7 action in cell death and mediator processing (e.g., pro-interleukin-1 cleavage by the inflammasome), but the contribution of P2X7 to transcriptional regulation is less well defined. In this review, we will focus on the growing evidence for the importance of nucleotide-mediated gene expression, we will highlight several animal model, human genetic, and clinical studies that support P2X7 as a therapeutic target, and we will discuss the latest developments in anti-P2X7 clinical trials. PMID:21298493

  2. The P2X7 receptor antagonist Brilliant Blue G attenuates contralateral rotations in a rat model of Parkinsonism through a combined control of synaptotoxicity, neurotoxicity and gliosis.

    PubMed

    Carmo, Marta R S; Menezes, Ana Paula F; Nunes, Ana Carla L; Pliássova, Anna; Rolo, Anabela P; Palmeira, Carlos M; Cunha, Rodrigo A; Canas, Paula M; Andrade, Geanne M

    2014-06-01

    Parkinson's disease (PD) involves an initial loss of striatal dopaminergic terminals evolving into a degeneration of dopaminergic neurons in the substantia nigra (SN), which can be modeled by 6-hydroxydopamine (6-OHDA) administration. Since ATP is a danger signal acting through its P2X7 receptors (P2X7R), we now tested if a blood-brain barrier-permeable P2X7R antagonist, Brilliant Blue G (BBG), controlled the 6-OHDA-induced PD-like features in rats. BBG (45 mg/kg) attenuated the 6-OHDA-induced: 1) increase of contralateral rotations in the apomorphine test, an effect mimicked by another P2X7R antagonist A438079 applied intra-cerebroventricularly; 2) short-term memory impairment in the passive avoidance and cued version of the Morris Water maze; 3) reduction of dopamine content in the striatum and SN; 4) microgliosis and astrogliosis in the striatum. To grasp the mechanism of action of BBG, we used in vitro models exploring synaptotoxicity (striatal synaptosomes) and neurotoxicity (dopamine-differentiated neuroblastoma SH-SY5Y cells). P2X7R were present in striatal dopaminergic terminals, and BBG (100 nM) prevented the 6-OHDA-induced synaptosomal dysfunction. P2X7R were also co-localized with tyrosine hydroxylase in SH-SY5Y cells, where BBG (100 nM) attenuated the 6-OHDA-induced neurotoxicity. This suggests that P2X7R contribute to PD pathogenesis through a triple impact on synaptotoxicity, gliosis and neurotoxicity, highlighting the therapeutic potential of P2X7R antagonists in PD. PMID:24508709

  3. PI3K/Akt signaling pathway triggers P2X7 receptor expression as a pro-survival factor of neuroblastoma cells under limiting growth conditions

    PubMed Central

    Gómez-Villafuertes, Rosa; García-Huerta, Paula; Díaz-Hernández, Juan Ignacio; Miras-Portugal, Mª Teresa

    2015-01-01

    The expression of purinergic P2X7 receptor (P2X7R) in neuroblastoma cells is associated to accelerated growth rate, angiogenesis, metastasis and poor prognosis. Noticeably, P2X7R allows the survival of neuroblastoma cells under restrictive conditions, including serum and glucose deprivation. Previously we identified specificity protein 1 (Sp1) as the main factor involved in the transcriptional regulation of P2rx7 gene, reporting that serum withdrawal triggers the expression of P2X7R in Neuro-2a (N2a) neuroblastoma cell line. Here we demonstrate that PI3K/Akt pathway is crucial for the upregulation of P2X7R expression in serum-deprived neuroblastoma cells, circumstance that facilitates cell proliferation in the absence of trophic support. The effect exerted by PI3K/Akt is independent of both mTOR and GSK3, but requires the activation of EGF receptor (EGFR). Nuclear levels of Sp1 are strongly reduced by inhibition of PI3K/Akt pathway, and blockade of Sp1-dependent transcription with mithramycin A prevents upregulation of P2rx7 gene expression following serum withdrawal. Furthermore, atypical PKCζ plays a key role in the regulation of P2X7R expression by preventing phosphorylation and, consequently, activation of Akt. Altogether, these data indicate that activation of EGFR enhanced the expression of P2X7R in neuroblastoma cells lacking trophic support, being PI3K/Akt/PKCζ signaling pathway and Sp1 mediating this pro-survival outcome. PMID:26687764

  4. Wnt3a mitigates acute lung injury by reducing P2X7 receptor-mediated alveolar epithelial type I cell death

    PubMed Central

    Guo, Y; Mishra, A; Weng, T; Chintagari, N R; Wang, Y; Zhao, C; Huang, C; Liu, L

    2014-01-01

    Acute lung injury (ALI) is characterized by pulmonary endothelial and epithelial cell damage, and loss of the alveolar–capillary barrier. We have previously shown that P2X7 receptor (P2X7R), a cell death receptor, is specifically expressed in alveolar epithelial type I cells (AEC I). In this study, we hypothesized that P2X7R-mediated purinergic signaling and its interaction with Wnt/β-catenin signaling contributes to AEC I death. We examined the effect of P2X7R agonist 2′-3′-O-(4-benzoylbenzoyl)-ATP (BzATP) and Wnt agonist Wnt3a on AEC I death in vitro and in vivo. We also assessed the therapeutic potential of Wnt3a in a clinically relevant ALI model of intratracheal lipopolysaccharide (LPS) exposure in ventilated mice. We found that the activation of P2X7R by BzATP caused the death of AEC I by suppressing Wnt/β-catenin signaling through stimulating glycogen synthase kinase-3β (GSK-3β) and proteasome. On the other hand, the activation of Wnt/β-catenin signaling by Wnt3a, GSK-3β inhibitor, or proteasome inhibitor blocked the P2X7R-mediated cell death. More importantly, Wnt3a attenuated the AEC I damage caused by intratracheal instillation of BzATP in rats or LPS in ventilated mice. Our results suggest that Wnt3a overrides the effect of P2X7R on the Wnt/β-catenin signaling to prevent the AEC I death and restrict the severity of ALI. PMID:24922070

  5. PI3K/Akt signaling pathway triggers P2X7 receptor expression as a pro-survival factor of neuroblastoma cells under limiting growth conditions.

    PubMed

    Gómez-Villafuertes, Rosa; García-Huerta, Paula; Díaz-Hernández, Juan Ignacio; Miras-Portugal, M Teresa

    2015-01-01

    The expression of purinergic P2X7 receptor (P2X7R) in neuroblastoma cells is associated to accelerated growth rate, angiogenesis, metastasis and poor prognosis. Noticeably, P2X7R allows the survival of neuroblastoma cells under restrictive conditions, including serum and glucose deprivation. Previously we identified specificity protein 1 (Sp1) as the main factor involved in the transcriptional regulation of P2rx7 gene, reporting that serum withdrawal triggers the expression of P2X7R in Neuro-2a (N2a) neuroblastoma cell line. Here we demonstrate that PI3K/Akt pathway is crucial for the upregulation of P2X7R expression in serum-deprived neuroblastoma cells, circumstance that facilitates cell proliferation in the absence of trophic support. The effect exerted by PI3K/Akt is independent of both mTOR and GSK3, but requires the activation of EGF receptor (EGFR). Nuclear levels of Sp1 are strongly reduced by inhibition of PI3K/Akt pathway, and blockade of Sp1-dependent transcription with mithramycin A prevents upregulation of P2rx7 gene expression following serum withdrawal. Furthermore, atypical PKCζ plays a key role in the regulation of P2X7R expression by preventing phosphorylation and, consequently, activation of Akt. Altogether, these data indicate that activation of EGFR enhanced the expression of P2X7R in neuroblastoma cells lacking trophic support, being PI3K/Akt/PKCζ signaling pathway and Sp1 mediating this pro-survival outcome. PMID:26687764

  6. Pulmonary Infection with Hypervirulent Mycobacteria Reveals a Crucial Role for the P2X7 Receptor in Aggressive Forms of Tuberculosis

    PubMed Central

    Amaral, Eduardo P.; Ribeiro, Simone C. M.; Lanes, Verônica R.; Almeida, Fabrício M.; de Andrade, Marcelle R. M.; Bomfim, Caio Cesar Barbosa; Salles, Érika M.; Bortoluci, Karina R.; Coutinho-Silva, Robson; Hirata, Mario H.; Alvarez, José M.; Lasunskaia, Elena B.; D'Império-Lima, Maria Regina

    2014-01-01

    The purinergic P2X7 receptor (P2X7R) is a sensor of extracellular ATP, a damage-associated molecule that is released from necrotic cells and that induces pro-inflammatory cytokine production and cell death. To investigate whether the innate immune response to damage signals could contribute to the development of pulmonary necrotic lesions in severe forms of tuberculosis, disease progression was examined in C57BL/6 and P2X7R−/− mice that were intratracheally infected with highly virulent mycobacterial strains (Mycobacterium tuberculosis strain 1471 of the Beijing genotype family and Mycobacterium bovis strain MP287/03). The low-dose infection of C57BL/6 mice with bacteria of these strains caused the rapid development of extensive granulomatous pneumonia with necrotic areas, intense bacillus dissemination and anticipated animal death. In contrast, in P2X7R−/− mice, the lung pathology presented with moderate infiltrates of mononuclear leukocytes without visible signs of necrosis; the disease attenuation was accompanied by a delay in mortality. In vitro, the hypervirulent mycobacteria grew rapidly inside macrophages and induced death by a P2X7R-dependent mechanism that facilitated the release of bacilli. Furthermore, these bacteria were resistant to the protective mechanisms elicited in macrophages following extracellular ATP stimulation. Based on this study, we propose that the rapid intracellular growth of hypervirulent mycobacteria results in massive macrophage damage. The ATP released by damaged cells engages P2X7R and accelerates the necrotic death of infected macrophages and the release of bacilli. This vicious cycle exacerbates pneumonia and lung necrosis by promoting widespread cell destruction and bacillus dissemination. These findings suggest the use of drugs that have been designed to inhibit the P2X7R as a new therapeutic approach to treat the aggressive forms of tuberculosis. PMID:24991816

  7. Activation of P2X7 Receptor by ATP Plays an Important Role in Regulating Inflammatory Responses during Acute Viral Infection

    PubMed Central

    Lee, Benjamin H.; Hwang, David M.; Palaniyar, Nades; Grinstein, Sergio; Philpott, Dana J.; Hu, Jim

    2012-01-01

    Acute viral infection causes damages to the host due to uncontrolled viral replication but even replication deficient viral vectors can induce systemic inflammatory responses. Indeed, overactive host innate immune responses to viral vectors have led to devastating consequences. Macrophages are important innate immune cells that recognize viruses and induce inflammatory responses at the early stage of infection. However, tissue resident macrophages are not easily activated by the mere presence of virus suggesting that their activation requires additional signals from other cells in the tissue in order to trigger inflammatory responses. Previously, we have shown that the cross-talk between epithelial cells and macrophages generates synergistic inflammatory responses during adenoviral vector infection. Here, we investigated whether ATP is involved in the activation of macrophages to induce inflammatory responses during an acute adenoviral infection. Using a macrophage-epithelial cell co-culture system we demonstrated that ATP signaling through P2X7 receptor (P2X7R) is required for induction of inflammatory mediators. We also showed that ATP-P2X7R signaling regulates inflammasome activation as inhibition or deficiency of P2X7R as well as caspase-1 significantly reduced IL-1β secretion. Furthermore, we found that intranasal administration of replication deficient adenoviral vectors in mice caused a high mortality in wild-type mice with symptoms of acute respiratory distress syndrome but the mice deficient in P2X7R or caspase-1 showed increased survival. In addition, wild-type mice treated with apyrase or inhibitors of P2X7R or caspase-1 showed higher rates of survival. The improved survival in the P2X7R deficient mice correlated with diminished levels of IL-1β and IL-6 and reduced neutrophil infiltration in the early phase of infection. These results indicate that ATP, released during viral infection, is an important inflammatory regulator that activates the

  8. Enhanced binding capability of nuclear factor-κB with demethylated P2X3 receptor gene contributes to cancer pain in rats.

    PubMed

    Zhou, You-Lang; Jiang, Guo-Qin; Wei, Jinrong; Zhang, Hong-Hong; Chen, Wei; Zhu, Hongyan; Hu, Shufen; Jiang, Xinghong; Xu, Guang-Yin

    2015-10-01

    Nuclear factor-kappa B (NF-κB) signaling is implicated in both cancer development and inflammation processes. However, the roles and mechanisms of NF-κB signaling in the development of cancer-induced pain (CIP) remain unknown. This study was designed to investigate the roles of the p65 subunit of NF-κB in regulation of the purinergic receptor (P2X3R) plasticity in dorsal root ganglion (DRG) of CIP rats. We showed here that tumor cell injection produced mechanical and thermal hyperalgesia, and an enhanced body weight-bearing difference, which was correlated with an upregulation of p65 and P2X3R expression in lumber DRGs and a potentiation of ATP-evoked responses of tibia-innervating DRG neurons. Inhibition of NF-κB signaling using p65 inhibitor pyrrolidine dithiocarbamate, BAY-11-7082, or lentiviral-p65 short-hairpin RNA significantly attenuated CIP and reversed the activities of P2X3R. Interestingly, tumor cell injection led to a significant demethylation of CpG island in p2x3r gene promoter and enhanced ability of p65 to bind the promoter of p2x3r gene. Our findings suggest that upregulation of P2X3R expression was mediated by the enhanced binding capability of p65 with demethylated promoter of p2x3r gene, thus contributing to CIP. NF-κBp65 might be a potential target for treating CIP, a neuropathic pain generated by tumor cell-induced injury to nerves that innervate the skin. PMID:26049406

  9. Enhanced binding capability of nuclear factor-κB with demethylated P2X3 receptor gene contributes to cancer pain in rats

    PubMed Central

    Zhou, You-Lang; Jiang, Guo-Qin; Wei, Jinrong; Zhang, Hong-Hong; Chen, Wei; Zhu, Hongyan; Hu, Shufen; Jiang, Xinghong; Xu, Guang-Yin

    2015-01-01

    Abstract Nuclear factor-kappa B (NF-κB) signaling is implicated in both cancer development and inflammation processes. However, the roles and mechanisms of NF-κB signaling in the development of cancer-induced pain (CIP) remain unknown. This study was designed to investigate the roles of the p65 subunit of NF-κB in regulation of the purinergic receptor (P2X3R) plasticity in dorsal root ganglion (DRG) of CIP rats. We showed here that tumor cell injection produced mechanical and thermal hyperalgesia, and an enhanced body weight–bearing difference, which was correlated with an upregulation of p65 and P2X3R expression in lumber DRGs and a potentiation of ATP-evoked responses of tibia-innervating DRG neurons. Inhibition of NF-κB signaling using p65 inhibitor pyrrolidine dithiocarbamate, BAY-11-7082, or lentiviral-p65 short-hairpin RNA significantly attenuated CIP and reversed the activities of P2X3R. Interestingly, tumor cell injection led to a significant demethylation of CpG island in p2x3r gene promoter and enhanced ability of p65 to bind the promoter of p2x3r gene. Our findings suggest that upregulation of P2X3R expression was mediated by the enhanced binding capability of p65 with demethylated promoter of p2x3r gene, thus contributing to CIP. NF-κBp65 might be a potential target for treating CIP, a neuropathic pain generated by tumor cell–induced injury to nerves that innervate the skin. PMID:26049406

  10. Activation of calcium-insensitive phospholipase A(2) (iPLA(2)) by P2X(7) receptors in murine peritoneal macrophages.

    PubMed

    El Ouaaliti, M; Seil, M; Dehaye, J P

    2012-12-01

    Free fatty acid releases are triggered by PLA2 activation and are substrates for many enzymes such as cyclooxygenases. These reactions are responsible for the production of many prostaglandins implicated in the inflammation yet many purinergic receptors have been implicated in diseases characterised by chronic inflammation. The role of P2X receptors was evaluated in LPS-primed murine peritoneal macrophages which were labelled with either [(3)H]-oleic acid or [(3)H]-arachidonic acid. Ten μmolar thapsigargin and 1mM ATP stimulated the release of both unsaturated acids. ATP had no effect at 10 μM and ivermectin had no effect on the response to ATP. The response to ATP was inhibited by magnesium and was not observed with cells from P2X(7)(-/-) mice. The response to ATP was not affected by the removal of extracellular calcium and was inhibited by arachidonyltrifluoromethyl ketone and bromoenol lactone but not by pyrrophenone. The release of the [(3)H]-fatty acids by ATP and thapsigargin was diminished by PD-98058, an inhibitor of MEK-1. It was concluded that in LPS-primed macrophages, P2X(7) receptors, not P2X(4) receptors, activated an iPLA(2) and promoted the release of unsaturated fatty acids secondary to the activation of a kinase. This response might contribute to the inflammation provoked by extracellular ATP. PMID:23041292

  11. Genetic interaction of P2X7 receptor and VEGFR-2 polymorphisms identifies a favorable prognostic profile in prostate cancer patients

    PubMed Central

    Solini, Anna; Simeon, Vittorio; Derosa, Lisa; Orlandi, Paola; Rossi, Chiara; Fontana, Andrea; Galli, Luca; Di Desidero, Teresa; Fioravanti, Anna; Lucchesi, Sara; Coltelli, Luigi; Ginocchi, Laura; Allegrini, Giacomo; Danesi, Romano; Falcone, Alfredo; Bocci, Guido

    2015-01-01

    VEGFR-2 and P2X7 receptor (P2X7R) have been described to stimulate the angiogenesis and inflammatory processes of prostate cancer. The present study has been performed to investigate the genetic interactions among VEGFR-2 and P2X7R SNPs and their correlation with overall survival (OS) in a population of metastatic prostate cancer patients. Analyses were performed on germline DNA obtained from blood samples and SNPs were investigated by real-time PCR technique. The survival dimensionality reduction (SDR) methodology was applied to investigate the genetic interaction between SNPs. One hundred patients were enrolled. The SDR software provided two genetic interaction profiles consisting of the combination between specific VEGFR-2 (rs2071559, rs11133360) and P2X7R (rs3751143, rs208294) genotypes. The median OS was 126 months (95% CI, 115.94–152.96) and 65.65 months (95% CI, 52.95–76.53) for the favorable and the unfavorable genetic profile, respectively (p < 0.0001). The genetic statistical interaction between VEGFR-2 (rs2071559, rs11133360) and P2X7R (rs3751143, rs208294) genotypes may identify a population of prostate cancer patients with a better prognosis. PMID:26337470

  12. Genetic interaction of P2X7 receptor and VEGFR-2 polymorphisms identifies a favorable prognostic profile in prostate cancer patients.

    PubMed

    Solini, Anna; Simeon, Vittorio; Derosa, Lisa; Orlandi, Paola; Rossi, Chiara; Fontana, Andrea; Galli, Luca; Di Desidero, Teresa; Fioravanti, Anna; Lucchesi, Sara; Coltelli, Luigi; Ginocchi, Laura; Allegrini, Giacomo; Danesi, Romano; Falcone, Alfredo; Bocci, Guido

    2015-10-01

    VEGFR-2 and P2X7 receptor (P2X7R) have been described to stimulate the angiogenesis and inflammatory processes of prostate cancer. The present study has been performed to investigate the genetic interactions among VEGFR-2 and P2X7R SNPs and their correlation with overall survival (OS) in a population of metastatic prostate cancer patients. Analyses were performed on germline DNA obtained from blood samples and SNPs were investigated by real-time PCR technique. The survival dimensionality reduction (SDR) methodology was applied to investigate the genetic interaction between SNPs. One hundred patients were enrolled. The SDR software provided two genetic interaction profiles consisting of the combination between specific VEGFR-2 (rs2071559, rs11133360) and P2X7R (rs3751143, rs208294) genotypes. The median OS was 126 months (95% CI, 115.94-152.96) and 65.65 months (95% CI, 52.95-76.53) for the favorable and the unfavorable genetic profile, respectively (p < 0.0001). The genetic statistical interaction between VEGFR-2 (rs2071559, rs11133360) and P2X7R (rs3751143, rs208294) genotypes may identify a population of prostate cancer patients with a better prognosis. PMID:26337470

  13. P2X4 receptor in the dorsal horn partially contributes to brain-derived neurotrophic factor oversecretion and toll-like receptor-4 receptor activation associated with bone cancer pain.

    PubMed

    Jin, Xiao-Hong; Wang, Li-Na; Zuo, Jian-Ling; Yang, Jian-Ping; Liu, Si-Lan

    2014-12-01

    Previous studies have suggested that the microglial P2X7 purinoceptor is involved in the release of tumor necrosis factor-α (TNFα) following activation of toll-like receptor-4 (TLR4), which is associated with nociceptive behavior. In addition, this progress is evoked by the activation of the P2X4 purinoceptor (P2X4R). Although P2X4R is also localized within spinal microglia in the dorsal horn, little is known about its role in cancer-induced bone pain (CIBP), which is in some ways unique. With the present rat model of CIBP, we demonstrate a critical role of the microglial P2X4R in the enhanced nociceptive transmission, which is associated with TLR4 activation and secretion of brain-derived neurotrophic factor (BDNF) and TNFα in the dorsal horn. We assessed mechanical threshold and spontaneous pain of CIBP rats. Moreover, P2X4R small interfering RNA (siRNA) was administered intrathecally, and real-time PCR, Western blots, immunofluorescence histochemistry, and ELISA were used to detect the expression of P2X4R, TLR4, OX-42, phosphorylated-p38 MAPK (p-p38), BDNF, and TNFα. Compared with controls, intrathecal injection of P2X4R siRNA could prevent nociceptive behavior induced by ATP plus lipopolysaccharide and CIBP and reduce the expression of P2X4R, TLR4, p-p38, BDNF, and TNFα. In addition, the increase of BDNF protein in rat microglial cells depended on P2X4 receptor signaling, which is partially associated with TLR4 activation. The ability of microglial P2X4R to activate TLR4 in spinal cord leading to behavioral hypersensitivity and oversecretion of BDNF could provide an opportunity for the prevention and treatment of CIBP. PMID:24984884

  14. P2X7 Receptor Antagonism Attenuates the Intermittent Hypoxia-induced Spatial Deficits in a Murine Model of Sleep Apnea Via Inhibiting Neuroinflammation and Oxidative Stress

    PubMed Central

    Deng, Yan; Guo, Xue-Ling; Yuan, Xiao; Shang, Jin; Zhu, Die; Liu, Hui-Guo

    2015-01-01

    Background: The mechanism of the neural injury caused by chronic intermittent hypoxia (CIH) that characterizes obstructive sleep apnea syndrome (OSAS) is not clearly known. The purpose of this study was to investigate whether P2X7 receptor (P2X7R) is responsible for the CIH-induced neural injury and the possible pathway it involves. Methods: Eight-week-old male C57BL/6 mice were used. For each exposure time point, eight mice divided in room air (RA) and IH group were assigned to the study of P2X7R expression. Whereas in the 21 days-Brilliant Blue G (BBG, a selective P2X7R antagonist) study, 48 mice were randomly divided into CIH group, BBG-treated CIH group, RA group and BBG-treated RA group. The hippocampus P2X7R expression was determined by Western blotting and real-time polymerase chain reaction (PCR). The spatial learning was analyzed by Morris water maze. The nuclear factor kappa B (NFκB) and NADPH oxidase 2 (NOX2) expressions were analyzed by Western blotting. The expressions of tumor necrosis factor α, interleukin 1β (IL-β), IL-18, and IL-6 were measured by real-time PCR. The malondialdehyde and superoxide dismutase levels were detected by colorimetric method. Cell damage was evaluated by Hematoxylin and Eosin staining and Terminal Transferase dUTP Nick-end Labeling method. Results: The P2X7R mRNA was elevated and sustained after 3-day IH exposure and the P2X7R protein was elevated and sustained after 7-day IH exposure. In the BBG study, the CIH mice showed severer neuronal cell damage and poorer performance in the behavior test. The increased NFκB and NOX2 expressions along with the inflammation injury and oxidative stress were also observed in the CIH group. BBG alleviated CIH-induced neural injury and consequent functional deficits. Conclusions: The P2X7R antagonism attenuates the CIH-induced neuroinflammation, oxidative stress, and spatial deficits, demonstrating that the P2X7R is an important therapeutic target in the cognition deficits accompanied

  15. Effect of electroacupuncture on P2X3 receptor regulation in the peripheral and central nervous systems of rats with visceral pain caused by irritable bowel syndrome.

    PubMed

    Weng, Z J; Wu, L Y; Zhou, C L; Dou, C Z; Shi, Y; Liu, H R; Wu, H G

    2015-09-01

    The aim of this study is to investigate the role of the purinergic receptor P2X3 in the peripheral and central nervous systems during acupuncture treatment for the visceral pain of irritable bowel syndrome (IBS). A total of 24 8-day-old Sprague-Dawley (SD) neonatal male rats (SPF grade) were stimulated using colorectal distention (CRD) when the rats were awake. The modeling lasted for 2 weeks with one stimulation per day. After 6 weeks, the rats were randomly divided into three groups of eight each: (1) the normal group (NG, n = 8); (2) the model group (MG, n = 8); and (3) the model + electroacupuncture group (EA, n = 8) that received electroacupuncture at a needling depth of 5 mm at the Shangjuxu (ST37, bilateral) and Tianshu (ST25, bilateral) acupoints. The parameters of the Han's acupoint nerve stimulator (HANS) were as follows: sparse-dense wave with a frequency of 2/100 Hz, current of 2 mA, 20 min/stimulation, and one stimulation per day; the treatment was provided for seven consecutive days. At the sixth week after the treatment, the abdominal withdrawal reflex (AWR) score was determined; immunofluorescence and immunohistochemistry were used to measure the expression of the P2X3 receptor in myenteric plexus neurons, prefrontal cortex, and anterior cingulate cortex; and, a real-time PCR assay was performed to measure the expression of P2X3 messenger RNA (mRNA) in the dorsal root ganglion (DRG) and spinal cord. After stimulation with CRD, the expression levels of the P2X3 receptor in the inter-colonic myenteric plexus, DRG, spinal cord, prefrontal cortex, and anterior cingulate cortex were upregulated, and the sensitivity of the rats to IBS visceral pain was increased. Electroacupuncture (EA) could downregulate the expression of the P2X3 receptor and ease the sensitivity to visceral pain. The P2X3 receptor plays an important role in IBS visceral pain. The different levels of P2X3 in the peripheral enteric nervous system and central nervous system mediate the

  16. Voltage- and ATP-dependent structural rearrangements of the P2X2 receptor associated with the gating of the pore.

    PubMed

    Keceli, Batu; Kubo, Yoshihiro

    2014-11-01

    together, we demonstrated that structural rearrangements of the linker region of the P2X2 receptor channel are induced not only by ligand binding but also by membrane potential change. PMID:25172943

  17. Voltage- and ATP-dependent structural rearrangements of the P2X2 receptor associated with the gating of the pore

    PubMed Central

    Keceli, Batu; Kubo, Yoshihiro

    2014-01-01

    demonstrated that structural rearrangements of the linker region of the P2X2 receptor channel are induced not only by ligand binding but also by membrane potential change. PMID:25172943

  18. Protective effects of naringin against gp120-induced injury mediated by P2X7 receptors in BV2 microglial cells.

    PubMed

    Chen, Q; Hu, J; Qin, S S; Liu, C L; Wu, H; Wang, J R; Lu, X M; Wang, J; Chen, G Q; Liu, Y; Liu, B Y; Xu, C S; Liang, S D

    2016-01-01

    This study was aimed at exploring the effects of P2X7 receptors on gp120-induced injury and naringin's protective effects against gp120-induced injury in BV2 microglia. BV2 microglia injury model was established by gp120 treatment and MTS assay was used to verify whether naringin has a cell-protective effect against gp120-induced injury. Changes in P2X7 receptor expression were assayed using RT-PCR, qPCR, and western blot. Results showed that the ODs of the Ctrl, gp120, gp120+naringin, and gp120+BBG groups were 0.91 ± 0.10, 0.71 ± 0.09, 0.83 ± 0.10, and 0.83 ± 0.10, respectively. Compared to the control group, the gp120 group showed a significantly decreased cell survival rate. Cell survival rates of the gp120+naringin group increased significantly compared to those of the gp120 group, while no difference was observed when compared to the gp120+BBG group. The relative P2X7 mRNA expression levels in the Ctrl, gp120, gp120+naringin, and gp120+BBG groups were 0.73 ± 0.06, 1.05 ± 0.06, 0.78 ± 0.05, and 0.81 ± 0.04, respectively. The corresponding P2X7 protein expression levels were 0.46 ± 0.04, 0.79 ± 0.04, 0.38 ± 0.07, and 0.42 ± 0.06. P2X7 mRNA and protein expression in the gp120 group increased significantly compared to those in the control group, and declined in the gp120+naringin group compared to those in the gp120 group. Therefore, P2X7 receptors might be involved in gp120-induced injury in BV2 microglia, and naringin might play a protective role by inhibiting the up-regulated expression of P2X7 receptors. PMID:27323041

  19. P2X-mediated AMPA receptor internalization and synaptic depression is controlled by two CaMKII phosphorylation sites on GluA1 in hippocampal neurons.

    PubMed

    Pougnet, Johan-Till; Compans, Benjamin; Martinez, Audrey; Choquet, Daniel; Hosy, Eric; Boué-Grabot, Eric

    2016-01-01

    Plasticity at excitatory synapses can be induced either by synaptic release of glutamate or the release of gliotransmitters such as ATP. Recently, we showed that postsynaptic P2X2 receptors activated by ATP released from astrocytes downregulate synaptic AMPAR, providing a novel mechanism by which glial cells modulate synaptic activity. ATP- and lNMDA-induced depression in the CA1 region of the hippocampus are additive, suggesting distinct molecular pathways. AMPARs are homo-or hetero-tetramers composed of GluA1-A4. Here, we first show that P2X2-mediated AMPAR inhibition is dependent on the subunit composition of AMPAR. GluA3 homomers are insensitive and their presence in heteromers alters P2X-mediated inhibition. Using a mutational approach, we demonstrate that the two CaMKII phosphorylation sites S567 and S831 located in the cytoplasmic Loop1 and C-terminal tail of GluA1 subunits, respectively, are critical for P2X2-mediated AMPAR inhibition recorded from co-expressing Xenopus oocytes and removal of surface AMPAR at synapses of hippocampal neurons imaged by the super-resolution dSTORM technique. Finally, using phosphorylation site-specific antibodies, we show that P2X-induced depression in hippocampal slices produces a dephosphorylation of the GluA1 subunit at S567, contrary to NMDAR-mediated LTD. These findings indicate that GluA1 phosphorylation of S567 and S831 is critical for P2X2-mediated AMPAR internalization and ATP-driven synaptic depression. PMID:27624155

  20. FasL-triggered death of Jurkat cells requires caspase 8-induced, ATP-dependent cross-talk between Fas and the purinergic receptor P2X(7).

    PubMed

    Aguirre, Adam; Shoji, Kenji F; Sáez, Juan C; Henríquez, Mauricio; Quest, Andrew F G

    2013-02-01

    Fas ligation via the ligand FasL activates the caspase-8/caspase-3-dependent extrinsic death pathway. In so-called type II cells, an additional mechanism involving tBid-mediated caspase-9 activation is required to efficiently trigger cell death. Other pathways linking FasL-Fas interaction to activation of the intrinsic cell death pathway remain unknown. However, ATP release and subsequent activation of purinergic P2X(7) receptors (P2X(7)Rs) favors cell death in some cells. Here, we evaluated the possibility that ATP release downstream of caspase-8 via pannexin1 hemichannels (Panx1 HCs) and subsequent activation of P2X(7)Rs participate in FasL-stimulated cell death. Indeed, upon FasL stimulation, ATP was released from Jurkat cells in a time- and caspase-8-dependent manner. Fas and Panx1 HCs colocalized and inhibition of the latter, but not connexin hemichannels, reduced FasL-induced ATP release. Extracellular apyrase, which hydrolyzes ATP, reduced FasL-induced death. Also, oxidized-ATP or Brilliant Blue G, two P2X(7)R blockers, reduced FasL-induced caspase-9 activation and cell death. These results represent the first evidence indicating that the two death receptors, Fas and P2X(7)R connect functionally via caspase-8 and Panx1 HC-mediated ATP release to promote caspase-9/caspase-3-dependent cell death in lymphoid cells. Thus, a hitherto unsuspected route was uncovered connecting the extrinsic to the intrinsic pathway to amplify death signals emanating from the Fas receptor in type II cells. PMID:22806078

  1. P2X(7) receptor in the kidneys of diabetic rats submitted to aerobic training or to N-acetylcysteine supplementation [corrected].

    PubMed

    Rodrigues, Adelson M; Bergamaschi, Cassia T; Fernandes, Maria Jose S; Paredes-Gamero, Edgar J; Buri, Marcus V; Curi, Marcus V; Ferreira, Alice T; Araujo, Sergio R R; Punaro, Giovana R; Maciel, Fabiane R; Nogueira, Guilherme B; Higa, Elisa M S

    2014-01-01

    Previous studies in our laboratory showed that N-acetylcysteine supplementation or aerobic training reduced oxidative stress and the progression of diabetic nephropathy in rats. The P2X(7 receptor is up-regulated in pathological conditions, such as diabetes mellitus. This up-regulation is related to oxidative stress and induces tissue apoptosis or necrosis. The aim of the present study is to assess the role of P2X(7) receptor in the kidneys of diabetic rats submitted to aerobic training or N-acetylcysteine supplementation. Diabetes was induced in male Wistar rats by streptozotocin (60 mg/kg, i.v.) and the training was done on a treadmill; N-acetylcysteine was given in the drinking water (600 mg/L). By confocal microscopy, as compared to control, the kidneys of diabetic rats showed increased P2 × 7 receptor expression and a higher activation in response to 2'(3')-O-(4-benzoylbenzoyl) adenosine5'-triphosphate (specific agonist) and adenosine triphosphate (nonspecific agonist) (all p<0.05). All these alterations were reduced in diabetic rats treated with N-acetylcysteine, exercise or both. We also observed measured proteinuria and albuminuria (early marker of diabetic nephropathy) in DM groups. Lipoperoxidation was strongly correlated with P2X(7) receptor expression, which was also correlated to NO•, thus associating this receptor to oxidative stress and kidney lesion. We suggest that P2X(7) receptor inhibition associated with the maintenance of redox homeostasis could be useful as coadjuvant treatment to delay the progression of diabetic nephropathy. PMID:24940871

  2. The relationship between P2X4 and P2X7: a physiologically important interaction?

    PubMed

    Craigie, Eilidh; Birch, Rebecca E; Unwin, Robert J; Wildman, Scott S

    2013-01-01

    Purinergic signaling within the kidney is becoming an important focus in the study of renal health and disease. The effectors of ATP signaling, the P2Y and P2X receptors, are expressed to varying extents in and along the nephron. There are many studies demonstrating the importance of the P2Y2 receptor on kidney function, and other P2 receptors are now emerging as participants in renal regulation. The P2X4 receptor has been linked to epithelial sodium transport in the nephron and expression levels of the P2X7 receptor are up-regulated in certain pathophysiological states. P2X7 antagonism has been shown to ameliorate rodent models of DOCA salt-induced hypertension and P2X4 null mice are hypertensive. Interestingly, polymorphisms in the genetic loci of P2X4 and P2X7 have been linked to blood pressure variation in human studies. In addition to the increasing evidence linking these two P2X receptors to renal function and health, a number of studies link the two receptors in terms of physical associations between their subunits, demonstrated both in vitro and in vivo. This review will analyze the current literature regarding interactions between P2X4 and P2X7 and assess the potential impact of these with respect to renal function. PMID:23966951

  3. Evaluation of the expression and function of the P2X7 receptor and ART1 in human regulatory T-cell subsets.

    PubMed

    Cortés-Garcia, Juan D; López-López, Cintya; Cortez-Espinosa, Nancy; García-Hernández, Mariana H; Guzmán-Flores, Juan M; Layseca-Espinosa, Esther; Portales-Cervantes, Liliana; Portales-Pérez, Diana P

    2016-01-01

    Regulatory T cells that express CD39 (CD39+ Treg) exhibit specific immunomodulatory properties. Ectonucleotidase CD39 hydrolyses ATP and ADP. ATP is a ligand of the P2X7 receptor and induces the shedding of CD62L and apoptosis. However, the role of ATP in CD39+ Treg cells has not been defined. Furthermore, NAD can activate the P2X7 receptor via ADP-ribosyltransferase (ART) enzymes and cause cell depletion in murine models. We evaluated the expression and function of P2X7 and ART1 in CD39+ Treg and CD39- Treg cells in the presence or absence of ATP and NAD. We isolated peripheral blood mononuclear cells from healthy subjects and purified CD4+ T cells, CD4+ CD25+ T cells and CD4+ CD25+ CD39+ T cells. P2X7 and ART1 expression was assessed by flow cytometry and real-time PCR. Our results showed low P2X7 expression on CD39+ Treg cells and higher levels of ART1 expression in CD4+ CD39+ T cells than the other subtypes studied. Neither shedding of CD62L nor cell death of CD39+ Treg or CD39- Treg cells was observed by 1mM ATP or 60μM NAD. In contrast, P2Xs receptor-dependent proliferation with 300μM ATP, was inhibited by NAD in the different cell types analysed. The NAD proliferation-inhibition was increased with P2Xs and A2a agonist and was reversed with P2Xs and A2a antagonist, therefore NAD inhibits P2Xs-dependent proliferation and A2a activation. In conclusion, our results suggest that the altered function and expression of P2X7 and ART1 in the human CD39+ Treg or CD39- Treg cells could participate in the resistance against cell death induced by ATP or NAD. PMID:26307000

  4. P2X Receptor-Dependent Erythrocyte Damage by α-Hemolysin from Escherichia coli Triggers Phagocytosis by THP-1 Cells

    PubMed Central

    Fagerberg, Steen K.; Skals, Marianne; Leipziger, Jens; Praetorius, Helle A.

    2013-01-01

    The pore-forming exotoxin α-hemolysin from E. coli causes a significant volume reduction of human erythrocytes that precedes the ultimate swelling and lysis. This shrinkage results from activation of Ca2+-sensitive K+ (KCa3.1) and Cl− channels (TMEM16A) and reduced functions of either of these channels potentiate the HlyA-induced hemolysis. This means that Ca2+-dependent activation of KCa3.1 and TMEM16A protects the cells against early hemolysis. Simultaneous to the HlyA-induced shrinkage, the erythrocytes show increased exposure of phosphatidylserine (PS) in the outer plasma membrane leaflet, which is known to be a keen trigger for phagocytosis. We hypothesize that exposure to HlyA elicits removal of the damaged erythrocytes by phagocytic cells. Cultured THP-1 cells as a model for erythrocytal phagocytosis was verified by a variety of methods, including live cell imaging. We consistently found the HlyA to very potently trigger phagocytosis of erythrocytes by THP-1 cells. The HlyA-induced phagocytosis was prevented by inhibition of KCa3.1, which is known to reduce PS-exposure in human erythrocytes subjected to both ionomycin and HlyA. Moreover, we show that P2X receptor inhibition, which prevents the cell damages caused by HlyA, also reduced that HlyA-induced PS-exposure and phagocytosis. Based on these results, we propose that erythrocytes, damaged by HlyA-insertion, are effectively cleared from the blood stream. This mechanism will potentially reduce the risk of intravascular hemolysis. PMID:23462688

  5. Nucleoside triphosphate diphosphohydrolase-1 ectonucleotidase is required for normal vas deferens contraction and male fertility through maintaining P2X1 receptor function.

    PubMed

    Kauffenstein, Gilles; Pelletier, Julie; Lavoie, Elise G; Kukulski, Filip; Martín-Satué, Mireia; Dufresne, Sébastien S; Frenette, Jérôme; Ribas Fürstenau, Cristina; Sereda, Michal J; Toutain, Bertrand; Henrion, Daniel; Sullivan, Robert; Vial, Catherine; Sévigny, Jean

    2014-10-10

    In this work, we report that Entpd1(-/-) mice, deficient for the ectonucleotidase nucleoside triphosphate diphosphohydrolase-1 (NTPDase1), produce smaller litters (27% reduction) compared with wild-type C57BL6 animals. This deficit is linked to reduced in vivo oocyte fertilization by Entpd1(-/-) males (61 ± 11% versus 88 ± 7% for Entpd1(+/+)). Normal epididymal sperm count, spermatozoa morphology, capacitation, and motility and reduced ejaculated sperm number (2.4 ± 0.5 versus 3.7 ± 0.4 million for Entpd1(+/+)) pointed to vas deferens dysfunction. NTPDase1 was localized by immunofluorescence in the tunica muscularis of the vas deferens. Its absence resulted in a major ATP hydrolysis deficiency, as observed in situ by histochemistry and in primary smooth muscle cell cultures. In vitro, Entpd1(-/-) vas deferens displayed an exacerbated contraction to ATP, a diminished response to its non-hydrolysable analog αβMeATP, and a reduced contraction to electrical field stimulation, suggesting altered P2X1 receptor function with a propensity to desensitize. This functional alteration was accompanied by a 3-fold decrease in P2X1 protein expression in Entpd1(-/-) vas deferens with no variation in mRNA levels. Accordingly, exogenous nucleotidase activity was required to fully preserve P2X1 receptor activation by ATP in vitro. Our study demonstrates that NTPDase1 is required to maintain normal P2X1 receptor functionality in the vas deferens and that its absence leads to impaired peristalsis, reduced spermatozoa concentration in the semen, and, eventually, reduced fertility. This suggests that alteration of NTPDase1 activity affects ejaculation efficacy and male fertility. This work may contribute to unveil a cause of infertility and open new therapeutic potentials. PMID:25160621

  6. P2X7 receptor activation downmodulates Na(+)-dependent high-affinity GABA and glutamate transport into rat brain cortex synaptosomes.

    PubMed

    Barros-Barbosa, A R; Lobo, M G; Ferreirinha, F; Correia-de-Sá, P; Cordeiro, J M

    2015-10-15

    Sodium-dependent high-affinity amino-acid transporters play crucial roles in terminating synaptic transmission in the central nervous system (CNS). However, there is lack of information about the mechanisms underlying the regulation of amino-acid transport by fast-acting neuromodulators, like ATP. Here, we investigated whether activation of the ATP-sensitive P2X7 receptor modulates Na(+)-dependent high-affinity γ-aminobutyric acid (GABA) and glutamate uptake into nerve terminals (synaptosomes) of the rat cerebral cortex. Radiolabeled neurotransmitter accumulation was evaluated by liquid scintillation spectrometry. The cell-permeant sodium-selective fluorescent indicator, SBFI-AM, was used to estimate Na(+) influx across plasma membrane. 2'(3')-O-(4-benzoylbenzoyl)ATP (BzATP, 3-300 μM), a prototypic P2X7 receptor agonist, concentration-dependently decreased [(3)H]GABA (14%) and [(14)C]glutamate (24%) uptake; BzATP decreased transport maximum velocity (Vmax) without affecting the Michaelis constant (Km) values. The selective P2X7 receptor antagonist, A-438079 (3 μM), prevented inhibition of [(3)H]GABA and [(14)C]glutamate uptake by BzATP (100 μM). The inhibitory effect of BzATP coincided with its ability to increase intracellular Na(+) and was mimicked by Na(+) ionophores, like gramicidin and monensin. Increases in intracellular Na(+) (with veratridine or ouabain) or substitution of extracellular Na(+) by N-methyl-D-glucamine (NMDG)(+) all decreased [(3)H]GABA and [(14)C]glutamate uptake and attenuated BzATP effects. Uptake inhibition by BzATP (100 μM) was also attenuated by calmidazolium, which selectively inhibits Na(+) currents through the P2X7 receptor pore. In conclusion, disruption of the Na(+) gradient by P2X7 receptor activation downmodulates high-affinity GABA and glutamate uptake into rat cortical synaptosomes. Interference with amino-acid transport efficacy may constitute a novel target for therapeutic management of cortical excitability. PMID

  7. Platelet Antistaphylococcal Responses Occur through P2X1 and P2Y12 Receptor-Induced Activation and Kinocidin Release▿

    PubMed Central

    Trier, Darin A.; Gank, Kimberly D.; Kupferwasser, Deborah; Yount, Nannette Y.; French, William J.; Michelson, Alan D.; Kupferwasser, Leon I.; Xiong, Yan Q.; Bayer, Arnold S.; Yeaman, Michael R.

    2008-01-01

    Platelets (PLTs) act in antimicrobial host defense by releasing PLT microbicidal proteins (PMPs) or PLT kinocidins (PKs). Receptors mediating staphylocidal efficacy and PMP or PK release versus isogenic PMP-susceptible (ISP479C) and -resistant (ISP479R) Staphylococcus aureus strains were examined in vitro. Isolated PLTs were incubated with ISP479C or ISP479R (PLT/S. aureus ratio range, 1:1 to 10,000:1) in the presence or absence of a panel of PLT inhibitors, including P2X and P2Y receptor antagonists of increasingly narrow specificity, and PLT adhesion receptors (CD41, CD42b, and CD62P). PLT-to-S. aureus exposure ratios of ≥10:1 yielded significant reductions in the viability of both strains. Results from reversed-phase high-performance liquid chromatography indicated that staphylocidal PLT releasates contained PMPs and PKs. At ratios below 10:1, the PLT antistaphylococcal efficacy relative to the intrinsic S. aureus PMP-susceptible or -resistant phenotype diminished. Apyrase (an agent of ADP degradation), suramin (a general P2 receptor antagonist), pyridoxal 5′-phosphonucleotide derivative (a specific P2X1 antagonist), and cangrelor (a specific P2Y12 antagonist) mitigated the PLT staphylocidal response against both strains, correlating with reduced levels of PMP and PK release. Specific inhibition occurred in the presence and absence of homologous plasma. The antagonism of the thromboxane A2, cyclooxygenase-1/cyclooxygenase-2, or phospholipase C pathway or the hindrance of surface adhesion receptors failed to impede PLT anti-S. aureus responses. These results suggest a multifactorial PLT anti-S. aureus response mechanism involving (i) a PLT-to-S. aureus ratio sufficient for activation; (ii) the ensuing degranulation of PMPs, PKs, ADP, and/or ATP; (iii) the activation of P2X1/P2Y12 receptors on adjacent PLTs; and (iv) the recursive amplification of PMP and PK release from these PLTs. PMID:18824536

  8. A comparative analysis of the activity of ligands acting at P2X and P2Y receptor subtypes in models of neuropathic, acute and inflammatory pain

    PubMed Central

    Andó, RD; Méhész, B; Gyires, K; Illes, P; Sperlágh, B

    2010-01-01

    Background and purpose: This study was undertaken to compare the analgesic activity of antagonists acting at P2X1, P2X7, and P2Y12 receptors and agonists acting at P2Y1, P2Y2, P2Y4, and P2Y6 receptors in neuropathic, acute, and inflammatory pain. Experimental approach: The effect of the wide spectrum P2 receptor antagonist PPADS, the selective P2X7 receptor antagonist Brilliant Blue G (BBG), the P2X1 receptor antagonist (4,4′,4″,4-[carbonylbis(imino-5,1,3-benzenetriyl-bis(carbonylimino))]tetrakis-1,3-benzenedisulfonic acid, octasodium salt (NF449) and (8,8′-[carbonylbis(imino-3,1-phenylenecarbonylimino)]bis-1,3,5-naphthalene-trisulphonic acid, hexasodium salt (NF023), the P2Y12 receptor antagonist (2,2-dimethyl-propionic acid 3-(2-chloro-6-methylaminopurin-9-yl)-2-(2,2-dimethyl-propionyloxymethyl)-propylester (MRS2395), the selective P2Y1 receptor agonist ([[(1R,2R,3S,4R,5S)-4-[6-amino-2-(methylthio)-9H-purin-9-yl]-2,3-dihydroxybicyclo[3.1.0]hex-1-yl]methyl] diphosphoric acid mono ester trisodium salt (MRS2365), the P2Y2/P2Y4 agonist uridine-5′-triphosphate (UTP), and the P2Y4/P2Y6 agonist uridine-5′-diphosphate (UDP) were examined on mechanical allodynia in the Seltzer model of neuropathic pain, on acute thermal nociception, and on the inflammatory pain and oedema induced by complete Freund's adjuvant (CFA). Key results: MRS2365, MRS2395 and UTP, but not the other compounds, significantly alleviated mechanical allodynia in the neuropathic pain model, with the following rank order of minimal effective dose (mED) values: MRS2365 > MRS2395 > UTP. All compounds had a dose-dependent analgesic action in acute pain except BBG, which elicited hyperalgesia at a single dose. The rank order of mED values in acute pain was the following: MRS2365 > MRS2395 > NF449 > NF023 > UDP = UTP > PPADS. MRS2365 and MRS2395 had a profound, while BBG had a mild effect on inflammatory pain, with a following rank order of mED values: MRS2395 > MRS2365 > BBG. None of the tested

  9. Concomitant blockade of P2X-receptors and ecto-nucleotidases by P2-receptor antagonists: functional consequences in rat vas deferens.

    PubMed

    Bültmann, R; Trendelenburg, M; Tuluc, F; Wittenburg, H; Starke, K

    1999-04-01

    In order to assess the consequences of a concomitant blockade of P2X-receptors and ecto-nucleotidases, effects of 13 P2-receptor antagonists were investigated on contractions of the rat vas deferens elicited by alpha,beta-methylene ATP (alpha,beta-MeATP) and ATP and on the removal of ATP from the incubation medium by vas deferens tissue. Increasing concentrations of all antagonists reduced and finally abolished contractions elicited by alpha,beta-MeATP (3 microM), with IC50-values ranging from 1.1 to 100 microM. Pyridoxalphosphate-6-azophenyl-2',4'-disulphonate (PPADS), 6-azophenyl-4-amino-5-hydroxy-naphthalene-1,3-disulphonate (NH02), 4,4'-diisothiocyanatostilbene-2,2'-disulphonate (DIDS) and uniblue A also progressively reduced and finally abolished contractions elicited by ATP (1 mM). 8,8'-[Carbonylbis(imino-3, 1-phenylenecarbonyl-imino)]-bis-(1,3,5-naphthalenetrisulphonate ) (NF023), suramin, pyridoxalphosphate-6-azophenyl-2',5'-disulphonate (iso-PPADS), trypan blue and reactive blue 19, in contrast, caused only partial blockade, by 34-43% maximally; reactive blue 2 and reactive red 2 had no effect; and 6,6'-(1,1'-biphenyl-4,4'-diylbisazo)-bis-4-amino-5-hydroxy-naphtha lene-1,3-disulphonate (NH01) and Evans blue even enhanced the response to ATP. For antagonists causing full or partial inhibition, the IC50-values against ATP were close to those against alpha,beta-MeATP. All antagonists attenuated the removal of ATP, with IC25%-values ranging from 0.8 microM to >320 microM. The results confirm the frequent combination, in one antagonist molecule, of P2-receptor blockade and blockade of ecto-nucleotidases. This dual action underlies the effect of such compounds on contractions of the vas deferens elicited by ATP which, for certain substances (e.g., suramin, reactive blue 2), can be explained by a simple model in which the antagonist simultaneously blocks the degradation of ATP and a single contraction-mediating receptor (P2X1). Several observations, however, do

  10. Lipopolysaccharide induces alveolar macrophage necrosis via CD14 and the P2x7 receptor leading to Interleukin-1α release

    PubMed Central

    Dagvadorj, Jargalsaikhan; Shimada, Kenichi; Chen, Shuang; Jones, Heather D.; Tumurkhuu, Gantsetseg; Zhang, Wenxuan; Wawrowsky, Kolja A.; Crother, Timothy R.; Arditi, Moshe

    2015-01-01

    SUMMARY Acute lung injury (ALI) remains a serious health issue with little improvement in our understanding of the pathophysiology and therapeutic approaches. We investigated the mechanism that lipopolysaccharide (LPS) induces early neutrophil recruitment to lungs and increases pulmonary vascular permeability during ALI. Intratracheal LPS induced release of pro-interleukin-1α (IL-1α) from necrotic alveolar macrophages (AM), which activated endothelial cells (EC) to induce vascular leakage via loss of vascular endothelial (VE)-cadherin. LPS triggered the AM purinergic receptor P2X7(R) to induce Ca2+ influx and ATP depletion, which led to necrosis. P2X7R deficiency significantly reduced necrotic death of AM and release of pro-IL-1α into the lung. CD14 was required for LPS binding to P2X7R, as CD14 neutralization significantly diminished LPS induced necrotic death of AM and pro-IL-1α release. These results demonstrate a key role for pro-IL-1α from necrotic alveolar macrophages in LPS-mediated ALI, as a critical initiator of increased vascular permeability and early neutrophil infiltration. PMID:25862090

  11. Quantitative structure–activity relationship study of P2X7 receptor inhibitors using combination of principal component analysis and artificial intelligence methods

    PubMed Central

    Ahmadi, Mehdi; Shahlaei, Mohsen

    2015-01-01

    P2X7 antagonist activity for a set of 49 molecules of the P2X7 receptor antagonists, derivatives of purine, was modeled with the aid of chemometric and artificial intelligence techniques. The activity of these compounds was estimated by means of combination of principal component analysis (PCA), as a well-known data reduction method, genetic algorithm (GA), as a variable selection technique, and artificial neural network (ANN), as a non-linear modeling method. First, a linear regression, combined with PCA, (principal component regression) was operated to model the structure–activity relationships, and afterwards a combination of PCA and ANN algorithm was employed to accurately predict the biological activity of the P2X7 antagonist. PCA preserves as much of the information as possible contained in the original data set. Seven most important PC's to the studied activity were selected as the inputs of ANN box by an efficient variable selection method, GA. The best computational neural network model was a fully-connected, feed-forward model with 7−7−1 architecture. The developed ANN model was fully evaluated by different validation techniques, including internal and external validation, and chemical applicability domain. All validations showed that the constructed quantitative structure–activity relationship model suggested is robust and satisfactory. PMID:26600858

  12. Quantitative structure-activity relationship study of P2X7 receptor inhibitors using combination of principal component analysis and artificial intelligence methods.

    PubMed

    Ahmadi, Mehdi; Shahlaei, Mohsen

    2015-01-01

    P2X7 antagonist activity for a set of 49 molecules of the P2X7 receptor antagonists, derivatives of purine, was modeled with the aid of chemometric and artificial intelligence techniques. The activity of these compounds was estimated by means of combination of principal component analysis (PCA), as a well-known data reduction method, genetic algorithm (GA), as a variable selection technique, and artificial neural network (ANN), as a non-linear modeling method. First, a linear regression, combined with PCA, (principal component regression) was operated to model the structure-activity relationships, and afterwards a combination of PCA and ANN algorithm was employed to accurately predict the biological activity of the P2X7 antagonist. PCA preserves as much of the information as possible contained in the original data set. Seven most important PC's to the studied activity were selected as the inputs of ANN box by an efficient variable selection method, GA. The best computational neural network model was a fully-connected, feed-forward model with 7-7-1 architecture. The developed ANN model was fully evaluated by different validation techniques, including internal and external validation, and chemical applicability domain. All validations showed that the constructed quantitative structure-activity relationship model suggested is robust and satisfactory. PMID:26600858

  13. Calcium-dependent regulation of Rab activation and vesicle fusion by an intracellular P2X ion channel.

    PubMed

    Parkinson, Katie; Baines, Abigail E; Keller, Thomas; Gruenheit, Nicole; Bragg, Laricia; North, R Alan; Thompson, Christopher R L

    2014-01-01

    Rab GTPases play key roles in the delivery, docking and fusion of intracellular vesicles. However, the mechanism by which spatial and temporal regulation of Rab GTPase activity is controlled is poorly understood. Here we describe a mechanism by which localized calcium release through a vesicular ion channel controls Rab GTPase activity. We show that activation of P2XA, an intracellular ion channel localized to the Dictyostelium discoideum contractile vacuole system, results in calcium efflux required for downregulation of Rab11a activity and efficient vacuole fusion. Vacuole fusion and Rab11a downregulation require the activity of CnrF, an EF-hand-containing Rab GAP found in a complex with Rab11a and P2XA. CnrF Rab GAP activity for Rab11a is enhanced by the presence of calcium and the EF-hand domain. These findings suggest that P2XA activation results in vacuolar calcium release, which triggers activation of CnrF Rab GAP activity and subsequent downregulation of Rab11a to allow vacuole fusion. PMID:24335649

  14. Lymphocytes from P2X7-deficient mice exhibit enhanced P2X7 responses

    PubMed Central

    Taylor, Simon R. J.; Gonzalez-Begne, Mireya; Sojka, Dorothy K.; Richardson, Jill C.; Sheardown, Steven A.; Harrison, Stephen M.; Pusey, Charles D.; Tam, Frederick W. K.; Elliott, James I.

    2009-01-01

    The purinergic receptor P2X7 is expressed on immune cells, and its stimulation results in the release of IL-1β from macrophages. Its absence, as evidenced from the analysis of two independent strains of P2X7-deficient mice, results in reduced susceptibility to inflammatory disease, and the molecule is an important, potential therapeutic target in autoimmunity. However, P2X7 has also been detected in several neuronal cell types, although its function and even its presence in these cells are highly contested, with anti-P2X7 antibodies staining brain tissue from both strains of P2X7−/− mice identically to wild-type mice. It has therefore been suggested that neurons express a distinct “P2X7-like” protein that has similar antibody recognition epitopes to P2X7 and some properties of the genuine receptor. In this study, we show that whereas P2X7 activity is absent from macrophages and dendritic cells in P2X7−/− animals, T cells from one gene-deficient strain unexpectedly exhibit higher levels of P2X7 activity than that found in cells from control, unmanipulated C57BL/6 mice. A potential mechanism for this tissue-specific P2X7 expression in P2X7−/− animals is discussed, as is the implication that the immune and indeed neuronal functions of P2X7 may have been underestimated. PMID:19276178

  15. Sensitization by pulmonary reactive oxygen species of rat vagal lung C-fibers: the roles of the TRPV1, TRPA1, and P2X receptors.

    PubMed

    Ruan, Ting; Lin, Yu-Jung; Hsu, Tien-Huan; Lu, Shing-Hwa; Jow, Guey-Mei; Kou, Yu Ru

    2014-01-01

    Sensitization of vagal lung C-fibers (VLCFs) induced by mediators contributes to the pathogenesis of airway hypersensitivity, which is characterized by exaggerated sensory and reflex responses to stimulants. Reactive oxygen species (ROS) are mediators produced during airway inflammation. However, the role of ROS in VLCF-mediated airway hypersensitivity has remained elusive. Here, we report that inhalation of aerosolized 0.05% H2O2 for 90 s potentiated apneic responses to intravenous capsaicin (a TRPV1 receptor agonist), α,β-methylene-ATP (a P2X receptor agonist), and phenylbiguanide (a 5-HT3 receptor agonist) in anesthetized rats. The apneic responses to these three stimulants were abolished by vagatomy or by perivagal capsaicin treatment, a procedure that blocks the neural conduction of VLCFs. The potentiating effect of H2O2 on the apneic responses to these VLCF stimulants was prevented by catalase (an enzyme that degrades H2O2) and by dimethylthiourea (a hydroxyl radical scavenger). The potentiating effect of H2O2 on the apneic responses to capsaicin was attenuated by HC-030031 (a TRPA1 receptor antagonist) and by iso-pyridoxalphosphate-6-azophenyl-2',5'-disulphonate (a P2X receptor antagonist). The potentiating effect of H2O2 on the apneic responses to α,β-methylene-ATP was reduced by capsazepine (a TRPV1 receptor antagonist), and by HC-030031. The potentiating effect of H2O2 on the apneic responses to phenylbiguanide was totally abolished when all three antagonists were combined. Consistently, our electrophysiological studies revealed that airway delivery of aerosolized 0.05% H2O2 for 90 s potentiated the VLCF responses to intravenous capsaicin, α,β-methylene-ATP, and phenylbiguanide. The potentiating effect of H2O2 on the VLCF responses to phenylbiguanide was totally prevented when all antagonists were combined. Inhalation of 0.05% H2O2 indeed increased the level of ROS in the lungs. These results suggest that 1) increased lung ROS sensitizes VLCFs

  16. Silica nanoparticles activate purinergic signaling via P2X7 receptor in dendritic cells, leading to production of pro-inflammatory cytokines.

    PubMed

    Nakanishi, Kana; Tsukimoto, Mitsutoshi; Tanuma, Sei-Ichi; Takeda, Ken; Kojima, Shuji

    2016-09-01

    We examined the mechanism of SNP-mediated stimulation of IL-1β and IL-18 production via P2R-mediated pathways in mouse bone marrow dendritic cells (mBMDCs). Examination of uptake of SNPs with diameters of 30, 70, and 300nm (SNP30, SNP70, and SNP300, respectively) by lipopolysaccharide-matured mBMDCs revealed that significant uptake of SNP30 occurred within as short a time as 1h. Production of IL-1β and IL-18 by cells exposed to SNPs increased dose-dependently, and was highest in cells exposed to SNP30. The SNP30-induced cytokine production was significantly inhibited by ATPase (apyrase) and by P2X7 receptor antagonist (A438079). ATP release was also highest in SNP30-exposed cells. Treatment of mBMDCs with exogenous ATP induced release of high levels of IL-1β and IL-18, and this release was also significantly inhibited by apyrase and A438079. The order of effectiveness of the three SNPs for inducing intracellular reactive oxygen species (ROS) production accorded well with those of cytokine production and ATP release. ROS production was inhibited by diphenyleneiodonium chloride (DPI). SNPs, especially SNP30, activate purinergic signaling in matured mBMDCs by inducing ATP release via P2X7 receptor. ATP induces ROS production via NADPH oxidase, and ROS activate inflammasomes, leading to caspase-1-dependent processing of pro-cytokines and release of IL-1β and IL-18. PMID:27311643

  17. Activation of P2X7 and P2Y11 purinergic receptors inhibits migration and normalizes tumor-derived endothelial cells via cAMP signaling

    PubMed Central

    Avanzato, D.; Genova, T.; Fiorio Pla, A.; Bernardini, M.; Bianco, S.; Bussolati, B.; Mancardi, D.; Giraudo, E.; Maione, F.; Cassoni, P.; Castellano, I.; Munaron, L.

    2016-01-01

    Purinergic signaling is involved in inflammation and cancer. Extracellular ATP accumulates in tumor interstitium, reaching hundreds micromolar concentrations, but its functional role on tumor vasculature and endothelium is unknown. Here we show that high ATP doses (>20 μM) strongly inhibit migration of endothelial cells from human breast carcinoma (BTEC), but not of normal human microvascular EC. Lower doses (1–10 mm result ineffective. The anti-migratory activity is associated with cytoskeleton remodeling and is significantly prevented by hypoxia. Pharmacological and molecular evidences suggest a major role for P2X7R and P2Y11R in ATP-mediated inhibition of TEC migration: selective activation of these purinergic receptors by BzATP mimics the anti-migratory effect of ATP, which is in turn impaired by their pharmacological or molecular silencing. Downstream pathway includes calcium-dependent Adenilyl Cyclase 10 (AC10) recruitment, cAMP release and EPAC-1 activation. Notably, high ATP enhances TEC-mediated attraction of human pericytes, leading to a decrease of endothelial permeability, a hallmark of vessel normalization. Finally, we provide the first evidence of in vivo P2X7R expression in blood vessels of murine and human breast carcinoma. In conclusion, we have identified a purinergic pathway selectively acting as an antiangiogenic and normalizing signal for human tumor-derived vascular endothelium. PMID:27586846

  18. Activation of P2X7 and P2Y11 purinergic receptors inhibits migration and normalizes tumor-derived endothelial cells via cAMP signaling.

    PubMed

    Avanzato, D; Genova, T; Fiorio Pla, A; Bernardini, M; Bianco, S; Bussolati, B; Mancardi, D; Giraudo, E; Maione, F; Cassoni, P; Castellano, I; Munaron, L

    2016-01-01

    Purinergic signaling is involved in inflammation and cancer. Extracellular ATP accumulates in tumor interstitium, reaching hundreds micromolar concentrations, but its functional role on tumor vasculature and endothelium is unknown. Here we show that high ATP doses (>20 μM) strongly inhibit migration of endothelial cells from human breast carcinoma (BTEC), but not of normal human microvascular EC. Lower doses (1-10 mm result ineffective. The anti-migratory activity is associated with cytoskeleton remodeling and is significantly prevented by hypoxia. Pharmacological and molecular evidences suggest a major role for P2X7R and P2Y11R in ATP-mediated inhibition of TEC migration: selective activation of these purinergic receptors by BzATP mimics the anti-migratory effect of ATP, which is in turn impaired by their pharmacological or molecular silencing. Downstream pathway includes calcium-dependent Adenilyl Cyclase 10 (AC10) recruitment, cAMP release and EPAC-1 activation. Notably, high ATP enhances TEC-mediated attraction of human pericytes, leading to a decrease of endothelial permeability, a hallmark of vessel normalization. Finally, we provide the first evidence of in vivo P2X7R expression in blood vessels of murine and human breast carcinoma. In conclusion, we have identified a purinergic pathway selectively acting as an antiangiogenic and normalizing signal for human tumor-derived vascular endothelium. PMID:27586846

  19. P2X6 Knockout Mice Exhibit Normal Electrolyte Homeostasis

    PubMed Central

    Viering, Daan H. H. M.; Bos, Caro; Bindels, René J. M.; Hoenderop, Joost G. J.

    2016-01-01

    ATP-mediated signaling is an important regulator of electrolyte transport in the kidney. The purinergic cation channel P2X6 has been previously localized to the distal convoluted tubule (DCT), a nephron segment important for Mg2+ and Na+ reabsorption, but its role in ion transport remains unknown. In this study, P2x6 knockout (P2x6-/-) mice were generated to investigate the role of P2X6 in renal electrolyte transport. The P2x6-/- animals displayed a normal phenotype and did not differ physiologically from wild type mice. Differences in serum concentration and 24-hrs urine excretion of Na+, K+, Mg2+ and Ca2+ were not detected between P2x6+/+, P2x6+/- and P2x6-/- mice. Quantitative PCR was applied to examine potential compensatory changes in renal expression levels of other P2x subunits and electrolyte transporters, including P2x1-5, P2x7, Trpm6, Ncc, Egf, Cldn16, Scnn1, Slc12a3, Slc41a1, Slc41a3, Cnnm2, Kcnj10 and Fxyd2. Additionally, protein levels of P2X2 and P2X4 were assessed in P2x6+/+ and P2x6-/- mouse kidneys. However, significant changes in expression were not detected. Furthermore, no compensatory changes in gene expression could be demonstrated in heart material isolated from P2x6-/- mice. Except for a significant (P<0.05) upregulation of P2x2 in the heart of P2x6-/- mice compared to the P2x6+/+ mice. Thus, our data suggests that purinergic signaling via P2X6 is not significantly involved in the regulation of renal electrolyte handling under normal physiological conditions. PMID:27254077

  20. Deletion of P2X7 attenuates hyperoxia-induced acute lung injury via inflammasome suppression.

    PubMed

    Galam, Lakshmi; Rajan, Ashna; Failla, Athena; Soundararajan, Ramani; Lockey, Richard F; Kolliputi, Narasaiah

    2016-03-15

    Increasing evidence shows that hyperoxia is a serious complication of oxygen therapy in acutely ill patients that causes excessive production of free radicals leading to hyperoxia-induced acute lung injury (HALI). Our previous studies have shown that P2X7 receptor activation is required for inflammasome activation during HALI. However, the role of P2X7 in HALI is unclear. The main aim of this study was to determine the effect of P2X7 receptor gene deletion on HALI. Wild-type (WT) and P2X7 knockout (P2X7 KO) mice were exposed to 100% O2 for 72 h. P2X7 KO mice treated with hyperoxia had enhanced survival in 100% O2 compared with the WT mice. Hyperoxia-induced recruitment of inflammatory cells and elevation of IL-1β, TNF-α, monocyte chemoattractant protein-1, and IL-6 levels were attenuated in P2X7 KO mice. P2X7 deletion decreased lung edema and alveolar protein content, which are associated with enhanced alveolar fluid clearance. In addition, activation of the inflammasome was suppressed in P2X7-deficient alveolar macrophages and was associated with suppression of IL-1β release. Furthermore, P2X7-deficient alveolar macrophage in type II alveolar epithelial cells (AECs) coculture model abolished protein permeability across mouse type II AEC monolayers. Deletion of P2X7 does not lead to a decrease in epithelial sodium channel expression in cocultures of alveolar macrophages and type II AECs. Taken together, these findings show that deletion of P2X7 is a protective factor and therapeutic target for the amelioration of hyperoxia-induced lung injury. PMID:26747786

  1. The nicotinic α6 subunit gene determines variability in chronic pain sensitivity via cross-inhibition of P2X2/3 receptors.

    PubMed

    Wieskopf, Jeffrey S; Mathur, Jayanti; Limapichat, Walrati; Post, Michael R; Al-Qazzaz, Mona; Sorge, Robert E; Martin, Loren J; Zaykin, Dmitri V; Smith, Shad B; Freitas, Kelen; Austin, Jean-Sebastien; Dai, Feng; Zhang, Jie; Marcovitz, Jaclyn; Tuttle, Alexander H; Slepian, Peter M; Clarke, Sarah; Drenan, Ryan M; Janes, Jeff; Al Sharari, Shakir; Segall, Samantha K; Aasvang, Eske K; Lai, Weike; Bittner, Reinhard; Richards, Christopher I; Slade, Gary D; Kehlet, Henrik; Walker, John; Maskos, Uwe; Changeux, Jean-Pierre; Devor, Marshall; Maixner, William; Diatchenko, Luda; Belfer, Inna; Dougherty, Dennis A; Su, Andrew I; Lummis, Sarah C R; Imad Damaj, M; Lester, Henry A; Patapoutian, Ardem; Mogil, Jeffrey S

    2015-05-13

    Chronic pain is a highly prevalent and poorly managed human health problem. We used microarray-based expression genomics in 25 inbred mouse strains to identify dorsal root ganglion (DRG)-expressed genetic contributors to mechanical allodynia, a prominent symptom of chronic pain. We identified expression levels of Chrna6, which encodes the α6 subunit of the nicotinic acetylcholine receptor (nAChR), as highly associated with allodynia. We confirmed the importance of α6* (α6-containing) nAChRs by analyzing both gain- and loss-of-function mutants. We find that mechanical allodynia associated with neuropathic and inflammatory injuries is significantly altered in α6* mutants, and that α6* but not α4* nicotinic receptors are absolutely required for peripheral and/or spinal nicotine analgesia. Furthermore, we show that Chrna6's role in analgesia is at least partially due to direct interaction and cross-inhibition of α6* nAChRs with P2X2/3 receptors in DRG nociceptors. Finally, we establish the relevance of our results to humans by the observation of genetic association in patients suffering from chronic postsurgical and temporomandibular pain. PMID:25972004

  2. The Nicotinic α6 Subunit Gene Determines Variability in Chronic Pain Sensitivity via Cross-inhibition of P2X2/3 Receptors

    PubMed Central

    Wieskopf, Jeffrey S.; Mathur, Jayanti; Limapichat, Walrati; Post, Michael R.; Al-Qazzaz, Mona; Sorge, Robert E.; Martin, Loren J.; Zaykin, Dmitri V.; Smith, Shad B.; Freitas, Kelen; Austin, Jean-Sebastien; Dai, Feng; Zhang, Jie; Marcovitz, Jaclyn; Tuttle, Alexander H.; Slepian, Peter M.; Clarke, Sarah; Drenan, Ryan M.; Janes, Jeff; Sharari, Shakir Al; Segall, Samantha K.; Aasvang, Eske K.; Lai, Weike; Bittner, Reinhard; Richards, Christopher I.; Slade, Gary D.; Kehlet, Henrik; Walker, John; Maskos, Uwe; Changeux, Jean-Pierre; Devor, Marshall; Maixner, William; Diatchenko, Luda; Belfer, Inna; Dougherty, Dennis A.; Su, Andrew I.; Lummis, Sarah C.R.; Damaj, M. Imad; Lester, Henry A.; Patapoutian, Ardem; Mogil, Jeffrey S.

    2016-01-01

    Chronic pain is a highly prevalent and poorly managed human health problem. We used microarray-based expression genomics in 25 inbred mouse strains to identify dorsal root ganglion (DRG)-expressed genetic contributors to mechanical allodynia, a prominent symptom of chronic pain. We identified expression levels of Chrna6, which encodes the α6 subunit of the nicotinic acetylcholine receptor (nAChR), as highly associated with allodynia. We confirmed the importance of α6* (i.e., α6-containing) nAChRs by analyzing both gain- and loss-of-function mutants. We find that mechanical allodynia associated with neuropathic and inflammatory injuries is significantly altered in α6* mutants, and that α6* but not α4* nicotinic receptors are absolutely required for peripheral and/or spinal nicotine analgesia. Furthermore, we show that Chrna6’s role in analgesia is at least partially due to direct interaction and cross-inhibition of α6* nAChRs with P2X2/3 receptors in DRG nociceptors. Finally, we establish relevance of our results to humans by the observation of genetic association in patients suffering from chronic postsurgical pain and temporomandibular pain. PMID:25972004

  3. P2X7 Deficiency Attenuates Renal Injury in Experimental Glomerulonephritis

    PubMed Central

    Taylor, Simon R.J.; Turner, Clare M.; Elliott, James I.; McDaid, John; Hewitt, Reiko; Smith, Jennifer; Pickering, Matthew C.; Whitehouse, Darren L.; Cook, H. Terence; Burnstock, Geoffrey; Pusey, Charles D.; Unwin, Robert J.; Tam, Frederick W.K.

    2009-01-01

    The P2X7 receptor is a ligand-gated cation channel that is normally expressed by a variety of immune cells, including macrophages and lymphocytes. Because it leads to membrane blebbing, release of IL-1β, and cell death by apoptosis or necrosis, it is a potential therapeutic target for a variety of inflammatory diseases. Although the P2X7 receptor is usually not detectable in normal renal tissue, we previously reported increased expression of both mRNA and protein in mesangial cells and macrophages infiltrating the glomeruli in animal models of antibody-mediated glomerulonephritis. In this study, we used P2X7-knockout mice in the same experimental model of glomerulonephritis and found that P2X7 deficiency was significantly renoprotective compared with wild-type controls, evidenced by better renal function, a striking reduction in proteinuria, and decreased histologic glomerular injury. In addition, the selective P2X7 antagonist A-438079 prevented the development of antibody-mediated glomerulonephritis in rats. These results support a proinflammatory role for P2X7 in immune-mediated renal injury and suggest that the P2X7 receptor is a potential therapeutic target. PMID:19389853

  4. Comparative study of the P2X gene family in animals and plants.

    PubMed

    Hou, Zhuoran; Cao, Jun

    2016-06-01

    P2X receptors are ligand-gated ion channels that can bind with the adenosine triphosphate (ATP) and have diverse functional roles in neuropathic pain, inflammation, special sense, and so on. In this study, 180 putative P2X genes, including 176 members in 32 animal species and 4 members in 3 species of lower plants, were identified. These genes were divided into 13 groups, including 7 groups in vertebrates and 6 groups in invertebrates and lower plants, through phylogenetic analysis. Their gene organization and motif composition are conserved in most predicted P2X members, while group-specific features were also found. Moreover, synteny relationships of the putative P2X genes in vertebrates are conserved while simultaneously experiencing a series of gene insertion, inversion, and transposition. Recombination signals were detected in almost all of the vertebrates and invertebrates, suggesting that intragenic recombination may play a significant role in the evolution of P2X genes. Selection analysis also identified some positively selected sites that acted on the evolution of most of the predicted P2X proteins. The phenomenon of alternative splicing occurred commonly in the putative P2X genes of vertebrates. This article explored in depth the evolutional relationship among different subtypes of P2X genes in animal and plants and might serve as a solid foundation for deciphering their functions in further studies. PMID:26874702

  5. LncRNA NONRATT021972 siRNA attenuates P2X7 receptor expression and inflammatory cytokine production induced by combined high glucose and free fatty acids in PC12 cells.

    PubMed

    Xu, Hong; He, Luling; Liu, Changle; Tang, Lan; Xu, Yonghu; Xiong, Mengqi; Yang, Mei; Fan, Yongfang; Hu, Fangfang; Liu, Xingzi; Ding, Lu; Gao, Yun; Xu, Changshui; Li, Guilin; Liu, Shuangmei; Wu, Bing; Zou, Lifang; Liang, Shangdong

    2016-06-01

    Diabetic neuropathy (DNP) is a frequent chronic complication of diabetes mellitus with potentially life-threatening outcomes. High glucose and elevated free fatty acids (FFAs) have been recently recognized as major causes of nervous system damage in diabetes. Our previous study has indicated extracellular stimuli, such as high glucose and/or FFA stress, may activate the p38 mitogen-activated protein kinase (MAPK) signaling pathway and induce a p38 MAPK-dependent sensitization of the P2X7 receptor and release of inflammatory factors in PC12 cells, while the mechanisms underlying remain to be elucidated. Long noncoding RNAs (lncRNAs) play important roles in diverse biological processes, including activation of a series of pathway signalings. Here, we showed combined high D-glucose and FFAs (HGHF) induced an increment of lncRNA-NONRATT021972 (NONCODE ID, nc021972) in PC12 cells. Nc021972 small interference RNA (siRNA) alleviated HGHF-induced activation of p38 MAPK, expression of the P2X7 receptor, and [Ca(2+)]i increment upon P2X7 receptor activation. Further experiments showed that there existed a crosstalk between nc021972 and the p38 MAPK signaling pathway. Inhibition of p38 MAPK signaling decreased nc021972-induced expression of the P2X7 receptor and [Ca(2+)]i increment upon P2X7 receptor activation. Also, nc021972 siRNA inhibited HGHF-induced PC12 release of TNF-α and IL-6 and rescued decreased cell viability mediated by the P2X7 receptor. Therefore, inhibition of nc021972 may serve as a novel therapeutic strategy for diabetes complicated with nervous inflammatory diseases. PMID:26865268

  6. P2X7R activation drives distinct IL-1 responses in dendritic cells compared to macrophages

    PubMed Central

    Englezou, Pavlos C.; Rothwell, Simon W.; Ainscough, Joseph S.; Brough, David; Landsiedel, Robert; Verkhratsky, Alexei; Kimber, Ian; Dearman, Rebecca J.

    2015-01-01

    The P2X7R is a functionally distinct member of the P2X family of non-selective cation channels associated with rapid activation of the inflammasome complex and signalling interleukin (IL)-1β release in macrophages. The main focus of this investigation was to compare P2X7R-driven IL-1 production by primary murine bone marrow derived dendritic cells (BMDC) and macrophages (BMM). P2X7R expression in murine BMDC and BMM at both transcriptional (P2X7A variant) and protein levels was demonstrated. Priming with lipopolysaccharide (LPS) and receptor activation with adenosine triphosphate (ATP) resulted in markedly enhanced IL-1 (α and β) secretion in BMDC compared with BMM. In both cell types IL-1 production was profoundly inhibited with a P2X7R-specific inhibitor (A-740003) demonstrating that this release is predominantly a P2X7R-dependent process. These data also suggest that P2X7R and caspase-1 activation drive IL-1α release from BMDC. Both cell types expressed constitutively the gain-of-function P2X7K as well as the full P2X7A variant at equivalent levels. LPS priming reduced significantly levels of P2X7A but not P2X7K transcripts in both BMDC and BMM. P2X7R-induced pore formation, assessed by YO-PRO-1 dye uptake, was greater in BMDC, and these cells were protected from cell death. These data demonstrate that DC and macrophages display distinct patterns of cytokine regulation, particularly with respect to IL-1, as a consequence of cell-type specific differences in the physicochemical properties of the P2X7R. Understanding the cell-specific regulation of these cytokines is essential for manipulating such responses in health and disease. PMID:26068648

  7. Acid-sensing ion channel 3 or P2X2/3 is involved in the pain-like behavior under a high bone turnover state in ovariectomized mice.

    PubMed

    Kanaya, Kumiko; Iba, Kousuke; Abe, Yasuhisa; Dohke, Takayuki; Okazaki, Shunichiro; Matsumura, Tadaki; Yamashita, Toshihiko

    2016-04-01

    We have recently demonstrated that pathological changes leading to increased bone resorption by osteoclast activation are related to the induction of pain-like behavior in ovariectomized (OVX) mice. In addition, bisphosphonate and the antagonist of transient receptor potential vanilloid type 1 (TRPV1), an acid-sensing nociceptor, improved the threshold value of pain-like behaviors accompanying an improvement in the acidic environment in the bone tissue based on osteoclast inactivation. The aim of this study was to evaluate the effect of (i) an inhibitor of vacuolar H(+) -ATPase, known as an proton pump, (ii) an antagonist of acid-sensing ion channel (ASIC) 3, as another acid-sensing nociceptor, and (iii) the P2X2/3 receptor, as an ATP-ligand nociceptor, on pain-like behavior in OVX mice. This inhibitor and antagonists were found to improve the threshold value of pain-like behavior in OVX mice. These results indicated that the skeletal pain accompanying osteoporosis is possibly associated with the acidic microenvironment and increased ATP level caused by osteoclast activation under a high bone turnover state. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:566-573, 2016. PMID:26340235

  8. A fluorescent approach for identifying P2X1 ligands

    PubMed Central

    Ruepp, Marc-David; Brozik, James A.; de Esch, Iwan J.P.; Farndale, Richard W.; Murrell-Lagnado, Ruth D.; Thompson, Andrew J.

    2015-01-01

    There are no commercially available, small, receptor-specific P2X1 ligands. There are several synthetic derivatives of the natural agonist ATP and some structurally-complex antagonists including compounds such as PPADS, NTP-ATP, suramin and its derivatives (e.g. NF279, NF449). NF449 is the most potent and selective ligand, but potencies of many others are not particularly high and they can also act at other P2X, P2Y and non-purinergic receptors. While there is clearly scope for further work on P2X1 receptor pharmacology, screening can be difficult owing to rapid receptor desensitisation. To reduce desensitisation substitutions can be made within the N-terminus of the P2X1 receptor, but these could also affect ligand properties. An alternative is the use of fluorescent voltage-sensitive dyes that respond to membrane potential changes resulting from channel opening. Here we utilised this approach in conjunction with fragment-based drug-discovery. Using a single concentration (300 μM) we identified 46 novel leads from a library of 1443 fragments (hit rate = 3.2%). These hits were independently validated by measuring concentration-dependence with the same voltage-sensitive dye, and by visualising the competition of hits with an Alexa-647-ATP fluorophore using confocal microscopy; confocal yielded kon (1.142 × 106 M−1 s−1) and koff (0.136 s−1) for Alexa-647-ATP (Kd = 119 nM). The identified hit fragments had promising structural diversity. In summary, the measurement of functional responses using voltage-sensitive dyes was flexible and cost-effective because labelled competitors were not needed, effects were independent of a specific binding site, and both agonist and antagonist actions were probed in a single assay. The method is widely applicable and could be applied to all P2X family members, as well as other voltage-gated and ligand-gated ion channels. This article is part of the Special Issue entitled ‘Fluorescent Tools in Neuropharmacology

  9. P2X7 Receptor Inhibition Improves CD34 T-Cell Differentiation in HIV-Infected Immunological Nonresponders on c-ART.

    PubMed

    Menkova-Garnier, Inna; Hocini, Hakim; Foucat, Emile; Tisserand, Pascaline; Bourdery, Laure; Delaugerre, Constance; Benne, Clarisse; Lévy, Yves; Lelièvre, Jean-Daniel

    2016-04-01

    Peripheral CD4+ T-cell levels are not fully restored in a significant proportion of HIV+ individuals displaying long-term viral suppression on c-ART. These immunological nonresponders (INRs) have a higher risk of developing AIDS and non-AIDS events and a lower life expectancy than the general population, but the underlying mechanisms are not fully understood. We used an in vitro system to analyze the T- and B-cell potential of CD34+ hematopoietic progenitor cells. Comparisons of INRs with matched HIV+ patients with high CD4+ T-cell counts (immune responders (IRs)) revealed an impairment of the generation of T-cell progenitors, but not of B-cell progenitors, in INRs. This impairment resulted in the presence of smaller numbers of recent thymic emigrants (RTE) in the blood and lower peripheral CD4+ T-cell counts. We investigated the molecular pathways involved in lymphopoiesis, focusing particularly on T-cell fate specification (Notch pathway), survival (IL7R-IL7 axis) and death (Fas, P2X7, CD39/CD73). P2X7 expression was abnormally strong and there was no CD73 mRNA in the CD34+ cells of INRs, highlighting a role for the ATP pathway. This was confirmed by the demonstration that in vitro inhibition of the P2X7-mediated pathway restored the T-cell potential of CD34+ cells from INRs. Moreover, transcriptomic analysis revealed major differences in cell survival and death pathways between CD34+ cells from INRs and those from IRs. These findings pave the way for the use of complementary immunotherapies, such as P2X7 antagonists, to restore T-cell lymphopoiesis in INRs. PMID:27082982

  10. P2X7 Receptor Inhibition Improves CD34 T-Cell Differentiation in HIV-Infected Immunological Nonresponders on c-ART

    PubMed Central

    Menkova-Garnier, Inna; Hocini, Hakim; Foucat, Emile; Tisserand, Pascaline; Bourdery, Laure; Delaugerre, Constance; Benne, Clarisse; Lévy, Yves; Lelièvre, Jean-Daniel

    2016-01-01

    Peripheral CD4+ T-cell levels are not fully restored in a significant proportion of HIV+ individuals displaying long-term viral suppression on c-ART. These immunological nonresponders (INRs) have a higher risk of developing AIDS and non-AIDS events and a lower life expectancy than the general population, but the underlying mechanisms are not fully understood. We used an in vitro system to analyze the T- and B-cell potential of CD34+ hematopoietic progenitor cells. Comparisons of INRs with matched HIV+ patients with high CD4+ T-cell counts (immune responders (IRs)) revealed an impairment of the generation of T-cell progenitors, but not of B-cell progenitors, in INRs. This impairment resulted in the presence of smaller numbers of recent thymic emigrants (RTE) in the blood and lower peripheral CD4+ T-cell counts. We investigated the molecular pathways involved in lymphopoiesis, focusing particularly on T-cell fate specification (Notch pathway), survival (IL7R-IL7 axis) and death (Fas, P2X7, CD39/CD73). P2X7 expression was abnormally strong and there was no CD73 mRNA in the CD34+ cells of INRs, highlighting a role for the ATP pathway. This was confirmed by the demonstration that in vitro inhibition of the P2X7-mediated pathway restored the T-cell potential of CD34+ cells from INRs. Moreover, transcriptomic analysis revealed major differences in cell survival and death pathways between CD34+ cells from INRs and those from IRs. These findings pave the way for the use of complementary immunotherapies, such as P2X7 antagonists, to restore T-cell lymphopoiesis in INRs. PMID:27082982