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1
Coutinho-Silva, Robson, Lynn Stahl, Kwok-Kuen Cheung, Nathalia Enes de Campos, Carolina de Oliveira Souza, David M. Ojcius, and Geoffrey Burnstock. "P2X and P2Y purinergic receptors on human intestinal epithelial carcinoma cells: effects of extracellular nucleotides on apoptosis and cell proliferation." American Journal of Physiology-Gastrointestinal and Liver Physiology 288, no. 5 (May 2005): G1024—G1035. http://dx.doi.org/10.1152/ajpgi.00211.2004.
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Extracellular nucleotides interact with purinergic receptors, which regulate ion transport in a variety of epithelia. With the use of two different human epithelial carcinoma cell lines (HCT8 and Caco-2), we have shown by RT-PCR that the cells express mRNA for P2X1, P2X3, P2X4, P2X5, P2X6, P2X7, P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, and P2Y12 receptors. Protein expression for P2Y1 and P2Y2 receptors was also demonstrated immunohistochemically, and P2X receptor subtype protein was present in the following decreasing order: P2X4 > P2X7 > P2X1 > P2X3 > P2X6 > P2X5 >> P2X2. The functional presence of P2X7, P2Y1, P2Y2, and P2Y4 receptors was shown based on the effect of extracellular nucleotides on apoptosis or cell proliferation, and measurement of nucleotide-dependent calcium fluxes using a fluorometric imaging plate reader in the presence of different selective agonists and antagonists. ATP, at high concentrations, induced apoptosis through ligation of P2X7 and P2Y1 receptors; conversely, ATP, at lower concentrations, and UTP stimulated proliferation, probably acting via P2Y2 receptors. We therefore propose that stimulation or dysfunction of purinergic receptors may contribute at least partially to modulation of epithelial carcinoma cell proliferation and apoptosis.
2
Ruan, Huai-Zhen, Lori A. Birder, William C. de Groat, Changfeng Tai, James Roppolo, Charles A. Buffington, and Geoffrey Burnstock. "Localization of P2X and P2Y Receptors in Dorsal Root Ganglia of the Cat." Journal of Histochemistry & Cytochemistry 53, no. 10 (June 27, 2005): 1273–82. http://dx.doi.org/10.1369/jhc.4a6556.2005.
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The distribution of P2X and P2Y receptor subtypes in upper lumbosacral cat dorsal root ganglia (DRG) has been investigated using immunohistochemistry. Intensity of immunoreactivity for six P2X receptors (P2X5 receptors were immuno-negative) and the three P2Y receptors examined in cat DRG was in the order of P2Y2 = P2Y4>P2X3>P2X2 = P2X7>P2X6>P2X1 = P2X4>P2Y1. P2X3, P2Y2, and P2Y4 receptor polyclonal antibodies stained 33.8%, 35.3%, and 47.6% of DRG neurons, respectively. Most P2Y2, P2X1, P2X3, P2X4, and P2X6 receptor staining was detected in small- and medium-diameter neurons. However, P2Y4, P2X2, and P2X7 staining was present in large- and small-diameter neurons. Double-labeling immunohistochemistry showed that 90.8%, 32.1%, and 2.4% of P2X3 receptor-positive neurons coexpressed IB4, CGRP, and NF200, respectively; whereas 67.4%, 41.3%, and 39.1% of P2Y4 receptor-positive neurons coexpressed IB4, CGRP, and NF200, respectively. A total of 18.8%, 16.6%, and 63.5% of P2Y2 receptor-positive neurons also stained for IB4, CGRP, and NF200, respectively. Only 30% of DRG neurons in cat were P2X3-immunoreactive compared with 90% in rat and in mouse. A further difference was the low expression of P2Y1 receptors in cat DRG neurons compared with more than 80% of the neurons in rat. Many small-diameter neurons were NF200-positive in cat, again differing from rat and mouse.
3
Birder, L. A., H. Z. Ruan, B. Chopra, Z. Xiang, S. Barrick, C. A. Buffington, J. R. Roppolo, A. P. D. W. Ford, W. C. de Groat, and G. Burnstock. "Alterations in P2X and P2Y purinergic receptor expression in urinary bladder from normal cats and cats with interstitial cystitis." American Journal of Physiology-Renal Physiology 287, no. 5 (November 2004): F1084—F1091. http://dx.doi.org/10.1152/ajprenal.00118.2004.
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Purinergic mechanisms appear to be involved in motor as well as sensory functions in the urinary bladder. ATP released from efferent nerves excites bladder smooth muscle, whereas ATP released from urothelial cells can activate afferent nerves and urothelial cells. In the present study, we used immunohistochemical techniques to examine the distribution of purinoceptors in the urothelium, smooth muscle, and nerves of the normal cat urinary bladder as well as possible changes in the expression of these receptors in cats with a chronic painful bladder condition termed feline interstitial cystitis (FIC) in which ATP release from the urothelium is increased. In normal cats, a range of P2X (P2X1, P2X2, P2X3, P2X4, P2X5, P2X6, and P2X7) and P2Y (P2Y1, P2Y2, and P2Y4) receptor subtypes was expressed throughout the bladder urothelium. In FIC cats, there is a marked reduction in P2X1 and loss of P2Y2 receptor staining. Both P2X3 and P2Y4 are present in nerves in normal cat bladder, and no obvious differences in staining were detected in FIC. Smooth muscle in the normal bladder did not exhibit P2Y receptor staining but did exhibit P2X (P2X2, P2X1) staining. In the FIC bladder smooth muscle, there was a significant reduction in P2X1 expression. These findings raise the possibility that purinergic mechanisms in the urothelium and bladder smooth muscle are altered in FIC cats. Because the urothelial cells appear to have a sensory function in the bladder, it is possible that the plasticity in urothelial purinergic receptors is linked with the painful bladder symptoms in IC.
4
Lee, B. M., H. Jo, G. Park, Y. H. Kim, C. K. Park, S. J. Jung, G. Chung, and S. B. Oh. "Extracellular ATP Induces Calcium Signaling in Odontoblasts." Journal of Dental Research 96, no. 2 (October 2, 2016): 200–207. http://dx.doi.org/10.1177/0022034516671308.
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Odontoblasts form dentin at the outermost surface of tooth pulp. An increasing level of evidence in recent years, along with their locational advantage, implicates odontoblasts as a secondary role as sensory or immune cells. Extracellular adenosine triphosphate (ATP) is a well-characterized signaling molecule in the neuronal and immune systems, and its potential involvement in interodontoblast communications was recently demonstrated. In an effort to elaborate the ATP-mediated signaling pathway in odontoblasts, the current study performed single-cell reverse transcription polymerase chain reaction (RT-PCR) and immunofluorescent detection to investigate the expression of ATP receptors related to calcium signal in odontoblasts from incisal teeth of 8- to 10-wk-old rats, and demonstrated an in vitro response to ATP application via calcium imaging experiments. While whole tissue RT-PCR analysis detected P2Y2, P2Y4, and all 7 subtypes (P2X1 to P2X7) in tooth pulp, single-cell RT-PCR analysis of acutely isolated rat odontoblasts revealed P2Y2, P2Y4, P2X2, P2X4, P2X6, and P2X7 expression in only a subset (23% to 47%) of cells tested, with no evidence for P2X1, P2X3, and P2X5 expression. An increase of intracellular Ca2+ concentration in response to 100μM ATP, which was repeated after pretreatment of thapsigargin or under the Ca2+-free condition, suggested function of both ionotropic and metabotropic ATP receptors in odontoblasts. The enhancement of ATP-induced calcium response by ivermectin and inhibition by 5-(3-bromophenyl)-1,3-dihydro-2H-benzofuro[3,2-e]-1,4-diazepin-2-one (5-BDBD) confirmed a functional P2X4 subtype in odontoblasts. Positive calcium response to 2′,3′-O-(benzoyl-4-benzoyl)-ATP (BzATP) and negative response to α,β-methylene ATP suggested P2X2, P2X4, and P2X7 as functional subunits in rat odontoblasts. Single-cell RT-PCR analysis of the cells with confirmed calcium response and immunofluorescent detection further corroborated the expression of P2X4 and P2X7 in odontoblasts. Overall, this study demonstrated heterogeneous expression of calcium-related ATP receptor subtypes in subsets of individual odontoblasts, suggesting extracellular ATP as a potential signal mediator for odontoblastic functions.
5
Baines, Abigail, Katie Parkinson, Joan A. Sim, Laricia Bragg, Christopher R. L. Thompson, and R. Alan North. "Functional Properties of Five Dictyostelium discoideum P2X Receptors." Journal of Biological Chemistry 288, no. 29 (June 5, 2013): 20992–1000. http://dx.doi.org/10.1074/jbc.m112.445346.
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The Dictyostelium discoideum genome encodes five proteins that share weak sequence similarity with vertebrate P2X receptors. Unlike vertebrate P2X receptors, these proteins are not expressed on the surface of cells, but populate the tubules and bladders of the contractile vacuole. In this study, we expressed humanized cDNAs of P2XA, P2XB, P2XC, P2XD, and P2XE in human embryonic kidney cells and altered the ionic and proton environment in an attempt to reflect the situation in amoeba. Recording of whole-cell membrane currents showed that four receptors operated as ATP-gated channels (P2XA, P2XB, P2XD, and P2XE). At P2XA receptors, ATP was the only effective agonist of 17 structurally related putative ligands that were tested. Extracellular sodium, compared with potassium, strongly inhibited ATP responses in P2XB, P2XD, and P2XE receptors. Increasing the proton concentration (pH 6.2) accelerated desensitization at P2XA receptors and decreased currents at P2XD receptors, but increased the currents at P2XB and P2XE receptors. Dictyostelium lacking P2XA receptors showed impaired regulatory volume decrease in hypotonic solution. This phenotype was readily rescued by overexpression of P2XA and P2XD receptors, partially rescued by P2XB and P2XE receptors, and not rescued by P2XC receptors. The failure of the nonfunctional receptor P2XC to restore the regulatory volume decrease highlights the importance of ATP activation of P2X receptors for a normal response to hypo-osmotic shock, and the weak rescue by P2XB and P2XE receptors indicates that there is limited functional redundancy among Dictyostelium P2X receptors.
6
Ruan, Huai Zhen, Lori A. Birder, Zhenghua Xiang, Bikramjit Chopra, Tony Buffington, Changfeng Tai, James R. Roppolo, William C. de Groat, and Geoffrey Burnstock. "Expression of P2X and P2Y receptors in the intramural parasympathetic ganglia of the cat urinary bladder." American Journal of Physiology-Renal Physiology 290, no. 5 (May 2006): F1143—F1152. http://dx.doi.org/10.1152/ajprenal.00333.2005.
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The distribution and function of P2X and P2Y receptor subtypes were investigated on intact or cultured intramural ganglia of the cat urinary bladder by immunocytochemistry and calcium-imaging techniques, respectively. Neurons were labeled by all seven P2X receptor subtype antibodies and antibodies for P2Y2, P2Y4, P2Y6, and P2Y12 receptor subtypes with a staining intensity of immunoreactivity in the following order: P2X3=P2Y2=P2Y4=P2Y6=P2Y12>P2X1=P2X2=P2X4>P2X5=P2X6=P2X7. P2Y1 receptor antibodies labeled glial cells, but not neurons. P2X3 and P2Y4 polyclonal antibodies labeled ∼95 and 40% of neurons, respectively. Double staining showed that 100, 48.8, and 97.4% of P2X3 receptor-positive neurons coexpressed choline acetyl transferase (ChAT), nitric oxide synthase (NOS), and neurofilament 200 (NF200), respectively, whereas 100, 59.2, and 97.6% of P2Y4 receptor-positive neurons coexpressed ChAT, NOS, and NF200, respectively. Application of ATP, α,β-methylene ATP, and uridine triphosphate elevated intracellular Ca2+ concentration in a subpopulation of dissociated cultured cat intramural ganglia neurons, demonstrating the presence of functional P2Y4 and P2X3 receptors. This study indicates that P2X and P2Y receptor subtypes are expressed by cholinergic parasympathetic neurons innervating the urinary bladder. The neurons were also stained for NF200, usually regarded as a marker for large sensory neurons. These novel histochemical properties of cholinergic neurons in the cat bladder suggest that the parasympathetic pathways to the cat bladder may be modulated by complex purinergic synaptic mechanisms.
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Roberts, V. H. J., S. L. Greenwood, A. C. Elliott, C. P. Sibley, and L. H. Waters. "Purinergic receptors in human placenta: evidence for functionally active P2X4, P2X7, P2Y2, and P2Y6." American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 290, no. 5 (May 2006): R1374—R1386. http://dx.doi.org/10.1152/ajpregu.00612.2005.
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Appropriate regulation of ion transport by the human placental syncytiotrophoblast is important for fetal growth throughout pregnancy. In nonplacental tissues, ion transport can be modulated by extracellular nucleotides that raise intracellular calcium ([Ca2+]i) via activation of purinergic receptors. We tested the hypothesis that purinergic receptors are expressed by human placental cytotrophoblast cells and that their activation by extracellular nucleotides modulates ion (K+) efflux and [Ca2+]i. P2X/P2Y receptor agonists 5-bromouridine 5′-triphosphate (5-BrUTP), ADP, ATP, 2′,3′- O-(4-benzoyl-benzoyl)adenosine 5′-triphosphate (BzATP), and UTP stimulated 86Rb (K+ tracer) efflux from cultured cytotrophoblast cells at early (mononuclear) or later (multinucleate syncytiotrophoblast-like) stages of differentiation, with ATP and UTP particularly potent. 2-Methylthioadenosine 5′-triphosphate (2-MeS-ATP), and UDP elevated 86Rb efflux only from multinucleated cells. All agonists caused a significant peak and plateau increase in [Ca2+]i, although the magnitude of responses was variable. The effect of BzATP, UTP, and UDP in multinucleated cells was unaffected, and that of ATP partially inhibited, by removal of extracellular Ca2+, implicating P2Y receptor activation. mRNA encoding P2X1, P2X2, P2X4, and P2X7 and P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11 were identified in mono- and multinucleated cells, whereas P2X3 and P2X5 mRNA were absent from all samples. Western blot analysis revealed P2X4, P2X7, P2Y2, and P2Y6 protein in cytotrophoblast cells, but P2Y4 was not detected. On the basis of published agonist selectivity, the data indicate the presence of functionally active P2X4, P2X7, P2Y2, and P2Y6 receptors in cytotrophoblast cells. We propose that activation of these receptors, and subsequent elevation of [Ca2+]i, modulates syncytiotrophoblast homeostasis and/or maternofetal ion exchange in response to extracellular nucleotides.
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Nakamura, Ei'Ichiro, Yasuhito Uezono, Ken'Ichiro Narusawa, Izumi Shibuya, Yosuke Oishi, Masahiro Tanaka, Nobuyuki Yanagihara, Toshitaka Nakamura, and Futoshi Izumi. "ATP activates DNA synthesis by acting on P2X receptors in human osteoblast-like MG-63 cells." American Journal of Physiology-Cell Physiology 279, no. 2 (August 1, 2000): C510—C519. http://dx.doi.org/10.1152/ajpcell.2000.279.2.c510.
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In human osteoblast-like MG-63 cells, extracellular ATP increased [3H]thymidine incorporation and cell proliferation and synergistically enhanced platelet-derived growth factor- or insulin-like growth factor I-induced [3H]thymidine incorporation. ATP-induced [3H]thymidine incorporation was mimicked by the nonhydrolyzable ATP analogs adenosine 5′- O-(3-thiotriphosphate) and adenosine 5′-adenylylimidodiphosphate and was inhibited by the P2 purinoceptor antagonist suramin, suggesting involvement of P2 purinoceptors. The P2Y receptor agonist UTP and UDP and a P2Y receptor antagonist reactive blue 2 did not affect [3H]thymidine incorporation, whereas the P2X receptor antagonist pyridoxal phosphate-6-azophenyl-2′,4-disulfonic acid inhibited ATP-induced [3H]thymidine incorporation, suggesting that ATP-induced DNA synthesis was mediated by P2X receptors. RT-PCR analysis revealed that MG-63 cells expressed P2X4, P2X5, P2X6, and P2X7, but not P2X1, P2X2, and P2X3, receptors. In fura 2-loaded cells, not only ATP, but also UTP, increased intracellular Ca2+concentration, and inhibitors for several Ca2+-activated protein kinases had no effect on ATP-induced DNA synthesis, suggesting that an increase in intracellular Ca2+concentration is not indispensable for ATP-induced DNA synthesis. ATP increased mitogen-activated protein kinase activity in a Ca2+-independent manner and synergistically enhanced platelet-derived growth factor- or insulin-like growth factor I-induced kinase activity. Furthermore, the mitogen-activated protein kinase kinase inhibitor PD-98059 totally abolished ATP-induced DNA synthesis. We conclude that ATP increases DNA synthesis and enhances the proliferative effects of growth factors through P2X receptors by activating a mitogen-activated protein kinase pathway.
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Chen, Lin, Changlong Leng, Qin Ru, Qi Xiong, Mei Zhou, and Yuxiang Wu. "Retrograde Labeling of Different Distribution Features of DRG P2X2 and P2X3 Receptors in a Neuropathic Pain Rat Model." BioMed Research International 2020 (July 23, 2020): 1–15. http://dx.doi.org/10.1155/2020/9861459.
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The distributions of P2X subtypes during peripheral neuropathic pain conditions and their differential roles are not fully understood. To explore these characteristics, the lumbosacral dorsal root ganglion (DRG) in the chronic constriction injury (CCI) sciatic nerve rat model was studied. Retrograde trace labeling combined with immunofluorescence technology was applied to analyze the distribution of neuropathic nociceptive P2X1-6 receptors. Our results suggest that Fluoro-Gold (FG) retrograde trace labeling is an efficient method for studying lumbosacral DRG neurons in the CCI rat model, especially when the DRG neurons are divided into small, medium, and large subgroups. We found that neuropathic nociceptive lumbosacral DRG neurons (i.e., FG-positive cells) were significantly increased in medium DRG neurons, while they declined in the large DRG neurons in the CCI group. P2X3 receptors were markedly upregulated in medium while P2X2 receptors were significantly decreased in small FG-positive DRG neurons. There were no significant changes in other P2X receptors (including P2X1, P2X4, P2X5, and P2X6). We anticipate that P2X receptors modulate nociceptive sensitivity primarily through P2X3 subtypes that are upregulated in medium neuropathic nociceptive DRG neurons and/or via the downregulation of P2X2 cells in neuropathic nociceptive small DRG neurons.
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Ramirez, Angelina N., and Diana L. Kunze. "P2X purinergic receptor channel expression and function in bovine aortic endothelium." American Journal of Physiology-Heart and Circulatory Physiology 282, no. 6 (June 1, 2002): H2106—H2116. http://dx.doi.org/10.1152/ajpheart.00892.2001.
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We examined bovine aortic endothelial cells (BAECs) for the functional expression of P2X receptors, the ATP-gated cation channels. We identified the P2X subtypes present in BAECs using RT-PCR. mRNA was present for only three of seven family members: P2X4, P2X5, and P2X7. We then characterized agonist-activated currents in whole cell and outside-out patch recordings using 2-methyl-thio-ATP (MeSATP) as a P2X4 and P2X5 receptor agonist and 2′,3′- O-(4-benzoylbenzoyl)ATP (BzATP) as a P2X7 receptor agonist. MeSATP (10–20 μM) produced current with characteristics of P2X4 receptors. The current was an inwardly rectifying current, reversed near 0 mV, slowly desensitized, was not blocked by suramin (300 μM) or reactive blue (60 μM), and had a single channel conductance of 36 pS. BzATP (10–100 μM), on the other hand, activated a 9-pS channel with sustained activity in the continued presence of the agonist. BzATP-activated current was blocked by reactive blue (60 μM) and by suramin (∼50% block at 300 μM). We confirmed, by immunocytochemistry, the presence of P2X4 and P2X7 protein. The agonists failed, however, to induce significant uptake of the large molecule YO-PRO, indicating the lack of pore development that has been demonstrated for P2X7 and P2X4 in response to agonist in some cell types.
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Rivera, Ian, Shali Zhang, B. Scott Fuller, Brentan Edwards, Tsugio Seki, Mong-Heng Wang, Mario B. Marrero, and Edward W. Inscho. "P2 receptor regulation of [Ca2+]i in cultured mouse mesangial cells." American Journal of Physiology-Renal Physiology 292, no. 5 (May 2007): F1380—F1389. http://dx.doi.org/10.1152/ajprenal.00349.2006.
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Experiments were performed to establish the pharmacological profile of purinoceptors and to identify the signal transduction pathways responsible for increases in intracellular calcium concentration ([Ca2+]i) for cultured mouse mesangial cells. Mouse mesangial cells were loaded with fura 2 and examined using fluorescent spectrophotometry. Basal [Ca2+]i averaged 102 ± 2 nM ( n = 346). One hundred micromolar concentrations of ATP, ADP, 2′,3′-(benzoyl-4-benzoyl)-ATP (BzATP), ATP-γ-S, and UTP in normal Ca2+ medium evoked peak increases in [Ca2+]i of 866 ± 111, 236 ± 18, 316 ± 26, 427 ± 37, and 808 ± 73 nM, respectively. UDP or 2-methylthio-ATP (2MeSATP) failed to elicit significant increases in [Ca2+]i, whereas identical concentrations of adenosine, AMP, and α,β-methylene ATP (α,β-MeATP) had no detectable effect on [Ca2+]i. Removal of Ca2+ from the extracellular medium had no significant effect on the peak increase in [Ca2+]i induced by ATP, ADP, BzATP, ATP-γ-S, or UTP compared with normal Ca2+; however, Ca2+-free conditions did accelerate the rate of decline in [Ca2+]i in cells treated with ATP and UTP. [Ca2+]i was unaffected by membrane depolarization with 143 mM KCl. Western blot analysis for P2 receptors revealed expression of P2X2, P2X4, P2X7, P2Y2, and P2Y4 receptors. No evidence of P2X1 and P2X3 receptor expression was detected, whereas RT-PCR analysis reveals mRNA expression for P2X1, P2X2, P2X3, P2X4, P2X7, P2Y2, and P2Y4 receptors. These data indicate that receptor-specific P2 receptor activation increases [Ca2+]i by stimulating calcium influx from the extracellular medium and through mobilization of Ca2+ from intracellular stores in cultured mouse mesangial cells.
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Trang, Mira, Günther Schmalzing, Christa E. Müller, and Fritz Markwardt. "Dissection of P2X4 and P2X7 Receptor Current Components in BV-2 Microglia." International Journal of Molecular Sciences 21, no. 22 (November 11, 2020): 8489. http://dx.doi.org/10.3390/ijms21228489.
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Microglia cells represent the immune system of the central nervous system. They become activated by ATP released from damaged and inflamed tissue via purinergic receptors. Ionotropic purinergic P2X4 and P2X7 receptors have been shown to be involved in neurological inflammation and pain sensation. Whether the two receptors assemble exclusively as homotrimers or also as heterotrimers is still a matter of debate. We investigated the expression of P2X receptors in BV-2 microglia cells applying the whole-cell voltage-clamp technique. We dissected P2X4 and P2X7 receptor-mediated current components by using specific P2X4 and P2X7 receptor blockers and by their characteristic current kinetics. We found that P2X4 and P2X7 receptors are activated independently from each other, indicating that P2X4/P2X7 heteromers are not of functional significance in these cells. The pro-inflammatory mediators lipopolysaccharide and interferon γ, if applied in combination, upregulated P2X4, but not P2X7 receptor-dependent current components also arguing against phenotypically relevant heteromerization of P2X4 and P2X7 receptor subunits.
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Gomes, Dayane A., Zhilin Song, Wanida Stevens, and Celia D. Sladek. "Sustained stimulation of vasopressin and oxytocin release by ATP and phenylephrine requires recruitment of desensitization-resistant P2X purinergic receptors." American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 297, no. 4 (October 2009): R940—R949. http://dx.doi.org/10.1152/ajpregu.00358.2009.
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Coexposure of hypothalamo-neurohypophyseal system explants to ATP and phenylephrine [PE; an α1-adrenergic receptor (α1-AR) agonist] induces an extended elevation in vasopressin and oxytocin (VP/OT) release. New evidence is presented that this extended response is mediated by recruitment of desensitization-resistant ionotropic purinergic receptor subtypes (P2X-Rs): 1) Antagonists of the P2X2/3 and P2X7-Rs truncated the sustained VP/OT release induced by ATP+PE but did not alter the transient response to ATP alone. 2) The P2X2/3 and P2X7-R antagonists did not alter either ATP or ATP+PE-induced increases in [Ca2+]i. 3) P2X2/3 and P2X7-R agonists failed to elevate [Ca2+]i, while ATP-γ-S, an agonist for P2X2-Rs increased [Ca2+]iand induced a transient increase in VP/OT release. 4) A P2Y1-R antagonist did not prevent initiation of the synergistic, sustained stimulation of VP/OT release by ATP+PE but did reduce its duration. Thus, the desensitization-resistant P2X2/3 and P2X7-R subtypes are required for the sustained, synergistic hormone response to ATP+PE, while P2X2-Rs are responsible for the initial activation of Ca2+-influx by ATP and ATP stimulation of VP/OT release. Immunohistochemistry, coimmunoprecipitation, and Western blot analysis confirmed the presence of P2X2 and P2X3, P2X2/3, and P2X7-R protein, respectively in SON. These findings support the hypothesis that concurrent activation of P2X2-R and α1-AR induces calcium-driven recruitment of P2X2/3 and 7-Rs, allowing sustained activation of a homeostatic circuit. Recruitment of these receptors may provide sustained release of VP during dehydration and may be important for preventing hemorrhagic and septic shock.
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Layhadi, Janice A., and Samuel J. Fountain. "ATP-Evoked Intracellular Ca2+ Responses in M-CSF Differentiated Human Monocyte-Derived Macrophage are Mediated by P2X4 and P2Y11 Receptor Activation." International Journal of Molecular Sciences 20, no. 20 (October 15, 2019): 5113. http://dx.doi.org/10.3390/ijms20205113.
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Tissues differentially secrete multiple colony stimulating factors under conditions of homeostasis and inflammation, orientating recruited circulating monocytes to differentiate to macrophage with differing functional phenotypes. Here, we investigated ATP-evoked intracellular Ca2+ responses in human macrophage differentiated with macrophage colony-stimulating factor (M-CSF). Extracellular ATP evoked (EC50 13.3 ± 1.4 μM) robust biphasic intracellular Ca2+ responses that showed a dependency on both metabotropic (P2Y) and ionotropic (P2X) receptors. qRT-PCR and immunocytochemistry revealed the expression of P2Y1, P2Y2, P2Y6, P2Y11, P2Y13, P2X1, P2X4, P2X5, and P2X7. Pharmacological analysis revealed contribution of only P2X4 and P2Y11 to the Ca2+ response evoked by maximal ATP concentrations (100 µM). This study reveals the contribution of P2X4 and P2Y11 receptor activation to ATP-evoked intracellular Ca2+ responses, and makes comparison with macrophage differentiated using granulocyte colony-stimulating factor (GM-CSF).
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Gui, Yu, ZengYong Wang, XiaoRui Sun, Michael P. Walsh, Jing-Jing Li, Jie Gao, and Xi-Long Zheng. "Uridine adenosine tetraphosphate induces contraction of airway smooth muscle." American Journal of Physiology-Lung Cellular and Molecular Physiology 301, no. 5 (November 2011): L789—L794. http://dx.doi.org/10.1152/ajplung.00203.2011.
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Contraction of airway smooth muscle (ASM) plays an important role in the regulation of air flow and is potentially involved in the pathophysiology of certain respiratory diseases. Extracellular nucleotides regulate ASM contraction via purinergic receptors, but the signaling mechanisms involved are not fully understood. Uridine adenosine tetraphosphate (Up4A) contains both pyrimidine and purine moieties, which are known to potentially activate P2X and P2Y receptors. Both P2X and P2Y receptors have been identified in the lung, including airway epithelial cells and ASM. We report here a study of purinergic signaling in the respiratory system, with a focus on the effect of Up4A on ASM contraction. Up4A induced contraction of rat isolated trachea and extrapulmonary bronchi as well as human intrapulmonary bronchioles. Up4A-induced contraction was blocked by di-inosine pentaphosphate, a P2X antagonist, but not by suramin, a nonselective P2 antagonist. Up4A-induced contraction was also attenuated by α,β-methylene-ATP-mediated P2X receptor desensitization. Several P2X receptors were detected at the mRNA level: P2X1, P2X4, P2X6, and P2X7, and to a lesser extent P2X3. Furthermore, the Up4A response was inhibited by removal of extracellular Ca2+ and by the presence of the L-type Ca2+ channel blocker, nifedipine, or the Rho-associated kinase inhibitor, H1152. We conclude that Up4A stimulates ASM contraction, and the underlying signaling mechanism appears to involve P2X (most likely P2X1) receptors, extracellular Ca2+ entry via L-type Ca2+ channels, and Ca2+ sensitization through the RhoA/Rho-associated kinase pathway. This study will add to our understanding of the pathophysiological roles of extracellular nucleotides in the lung.
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Bujko, Kamila, Mateusz Adamiak, Arjun Thapa, Magdalena Kucia, Janina Ratajczak, and Mariusz Z. Ratajczak. "Novel Evidence That Extracellular Adenosine Triphosphate (ATP), As a Purinergic Signaling Mediator, Activates Mobilization By Engaging a P2X4 Ligand-Gated Cation Channel Receptor Expressed on the Surface of Hematopoietic and Innate Immunity Cells." Blood 134, Supplement_1 (November 13, 2019): 4472. http://dx.doi.org/10.1182/blood-2019-125858.
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Background . Adenosine triphosphate (ATP) is an important nucleotide involved in intracellular energy transfer, but it is also released from activated cells into the extracellular space as a crucial component of the purinergic signaling network. Purinergic signaling is an ancient form of extracellular signaling that is mediated by ATP and other extracellular nucleotides (EXNs). Purinergic receptors for EXNs are expressed on the surface of all cells in the body; are represented by several families of P1, P2X, and P2Y receptors; and are among the most abundant receptors in living organisms. Of all these receptors, the P2X receptor family is most highly specific for ATP signaling and consists of seven members (P2X1-7). We recently reported that by activating the P2X7 receptor ATP activates the Nlrp3 inflammasome in hematopoietic cells and significantly enhances G-CSF-induced mobilization of hematopoietic stem progenitor cells (HSPCs, Leukemia 2018, 32:1920-1931). Hypothesis. Since P2X7-KO mice still mobilize some HSPCs, we hypothesized that, in addition to the P2X7 receptor, other receptors from this family also play a role in the mobilization process.Materials and Methods. RT-PCR and FACS were employed to phenotype human and murine bone marrow (BM) mononuclear cells (MNCs) as well as sorted HSCs for expression of P2X receptors. Mobilization studies were performed in wild type mice in which the P2X4 receptor was blocked by the specific small-molecule inhibitor PSB12054 and in P2X7-KO animals. Following mobilization, we measured i) the total number of white blood cells (WBCs) and ii) the number of circulating clonogenic colony-forming unit granulocyte/macrophage (CFU-GM) progenitors and Sca-1+c-kit+lineage- (SKL) cells circulating in PB. We also evaluated activation of the Nlrp3 inflammasome as well as other inflammasomes expressed in hematopoietic cells, including Aim2, Nlrp1a, Nlrp1b, and Nlrc4. Results. We found that, of all the receptors of the P2X family, two members, P2X7 and P2X4, are highly expressed on the surface of murine and human MNCs and HSPCs (Figure 1a). Activation of both receptors on the surface of innate immunity CD11b+Gr-1+ cells resulted in activation of the Nlrp3, Nlrp1a, Nlrp1b, and Aim2 inflammasomes. Inhibition of the P2X4 receptor by a specific small-molecule inhibitor decreased G-CSF- and AMD3100-induced mobilization in wild type mice (Figure 1b). Interestingly, we observed differences during AMD3100 mobilization, namely that while P2X7-KO deficiency resulted in decreased G-CSF-induced mobilization and did not affect AMD3100 mobilization efficiency, P2X4 inhibition, by contrast, led to a profound decrease in both G-CSF- and AMD3100-induced mobilization efficiency. Conclusions. Our results further support an important role for purinergic signaling in the mobilization of HSPCs. We also demonstrated that P2X7 and P2X4 receptors, which are highly expressed on human and murine hematopoietic cells, are involved in G-CSF-induced mobilization by activating the Nlrp3, Nlrp1a, Nlrp1b and Aim2 inflammasomes. By contrast, AMD3100-induced mobilization requires expression of P2X4 but not P2X7 receptors, which implies the involvement of different mechanisms and indicates that P2X7 deficiency can be compensated by the P2X4 receptor. Currently, we are performing RNASeq studies to identify differences in the G-CSF- and AMD3100-induced pathways in the mobilization process. These investigations will shed more light on the molecular pathways involved in the egress of HSPCs from BM into PB and will help to design better mobilization protocols in cases of poor mobilizers. Figure 1 Disclosures No relevant conflicts of interest to declare.
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Yamamoto, Kimiko, Risa Korenaga, Akira Kamiya, Zhi Qi, Masahiro Sokabe, and Joji Ando. "P2X4 receptors mediate ATP-induced calcium influx in human vascular endothelial cells." American Journal of Physiology-Heart and Circulatory Physiology 279, no. 1 (July 1, 2000): H285—H292. http://dx.doi.org/10.1152/ajpheart.2000.279.1.h285.
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ATP induces Ca2+ influx across the cell membrane and activates release from intracellular Ca2+ pools in vascular endothelial cells (ECs). Ca2+ signaling leads to the modification of a variety of EC functions, including the production of vasoactive substances such as nitric oxide and prostacyclin. However, the molecular mechanisms for ATP-induced Ca2+ influx in ECs have not been thoroughly clarified. Here we demonstrate evidence that a P2X4receptor for an ATP-gated cation channel is predominantly expressed in human ECs and is involved in the ATP-induced Ca2+ influx. Northern blot analysis distinctly showed the expression of P2X4 mRNA in human ECs cultured from the umbilical vein, aorta, pulmonary artery, and skin microvessels. Competitive PCR revealed that P2X4 mRNA expression was much higher in ECs than was the expression of other subtypes, including P2X1, P2X3, P2X5, and P2X7. Treatment of ECs with antisense oligonucleotides designed to target the P2X4 receptor decreased the P2X4 mRNA and protein levels to ∼25% of control levels and markedly prevented the ATP-induced Ca2+ influx.
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Pippel, Anja, Michaela Stolz, Ronja Woltersdorf, Achim Kless, Günther Schmalzing, and Fritz Markwardt. "Localization of the gate and selectivity filter of the full-length P2X7 receptor." Proceedings of the National Academy of Sciences 114, no. 11 (February 24, 2017): E2156—E2165. http://dx.doi.org/10.1073/pnas.1610414114.
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The P2X7 receptor (P2X7R) belongs to the P2X family of ATP-gated cation channels. P2X7Rs are expressed in epithelial cells, leukocytes, and microglia, and they play important roles in immunological and inflammatory processes. P2X7Rs are obligate homotrimers, with each subunit having two transmembrane helices, TM1 and TM2. Structural and functional data regarding the P2X2 and P2X4 receptors indicate that the central trihelical TM2 bundle forms the intrinsic transmembrane channel of P2X receptors. Here, we studied the accessibility of single cysteines substituted along the pre-TM2 and TM2 helix (residues 327–357) of the P2X7R using as readouts (i) the covalent maleimide fluorescence accessibility of the surface-bound P2X7R and (ii) covalent modulation of macroscopic and single-channel currents using extracellularly and intracellularly applied methanethiosulfonate (MTS) reagents. We found that the channel opening extends from the pre-TM2 region through the outer half of the trihelical TM2 channel. Covalently adducted MTS ethylammonium+ (MTSEA+) strongly increased the probability that the channel was open by delaying channel closing of seven of eight responsive human P2X7R (hP2X7R) mutants. Structural modeling, as supported by experimental probing, suggested that resulting intraluminal hydrogen bonding interactions stabilize the open-channel state. The additional decrease in single-channel conductance by MTSEA+ in five of seven positions identified Y336, S339, L341C, Y343, and G345 as the narrowest part of the channel lumen. The gate and ion-selectivity filter of the P2X7R could be colocalized at and around residue S342. None of our results provided any evidence for dilation of the hP2X7R channel on sustained stimulation with ATP4−.
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Mutini, Carmela, Simonetta Falzoni, Davide Ferrari, Paola Chiozzi, Anna Morelli, O. Roberto Baricordi, Ginetta Collo, Paola Ricciardi-Castagnoli, and Francesco Di Virgilio. "Mouse Dendritic Cells Express the P2X7 Purinergic Receptor: Characterization and Possible Participation in Antigen Presentation." Journal of Immunology 163, no. 4 (August 15, 1999): 1958–65. http://dx.doi.org/10.4049/jimmunol.163.4.1958.
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Abstract Immune cells express P2 purinoceptors of the P2Y and P2X subtypes. In the present work, we show that three dendritic cell (DC) lines, D2SC/1, CB1, and FSDC, representative of immature DCs, express the P2X7 (formerly P2Z) receptor, as judged from RT-PCR amplification, reactivity to a specific antiserum, and pharmacological and functional evidence. Receptor expression is higher in FSDC cells, a cell line that is functionally more mature than D2SC/1 and CB1. From the wild-type DC population, we selected cell clones lacking the P2X7R (P2X7less). We also used a P2XR blocker, oxidized ATP, to irreversibly inhibit the P2X7R. Ability of P2X7less FSDCs or of oxidized ATP-inhibited FSDCs to stimulate Ag-specific TH lymphocytes was severely decreased although Ag endocytosis was minimally affected. During coculture with TH lymphocytes, wild-type FSDC secreted large amounts of IL-1β. Release of this cytokine was reduced in P2X7less DCs. These data show that DCs express the P2X7 purinoceptor and suggest a correlation between P2X7R expression and Ag-presenting activity.
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Cardoso, Andréia Machado, Maria Luiza Mukai Franciosi, and Adriana Wagner. "What is new in Purinergic Signaling and Cervical Cancer?" Cancer Research and Cellular Therapeutics 5, no. 2 (June 9, 2021): 01–03. http://dx.doi.org/10.31579/2640-1053/084.
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Since our publication “Purinergic Signaling and Tumor Microenvironment in Cervical Cancer” [1], in early 2020, there has been a significant change in purinergic signaling research. The Coronavirus disease 2019 (COVID-19) significantly impacted the prevention, diagnosis, and treatment of cervical cancer [2]. In that previous review, we had addressed the possibilities of purinergic signaling in the tumor microenvironment of this type of cancer [1]. The conclusions were: the extracellular medium of cervical cancer is rich in adenosine triphosphate (ATP) and adenosine [3, 4, 5]; ATP is a pro-inflammatory molecule that has an affinity for P2X2, P2X4, and P2X7 receptors [6]; this activation leads to apoptosis of the cells of the cervix [7]; P2X7 is still involved in stimulating factors that lead to mitogenic and angiogenic pathways [8]; there is a variant of P2X7 in cervical cancer cells, P2X7j, which decreases permeability and cell death [9, 10, 11]. The P2Y1, P2Y2, and P2Y6 receptors, in turn, have the effect of tumor progression [12]. The review also contributed to the understanding of adenosine, which would activate A2A receptors on T lymphocytes, which would promote a decrease in the proliferation and effector function of such cells
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Arkhipov, Sergey N., D’Anna L. Potter, Aron M. Geurts, and Tengis S. Pavlov. "Knockout of P2rx7 purinergic receptor attenuates cyst growth in a rat model of ARPKD." American Journal of Physiology-Renal Physiology 317, no. 6 (December 1, 2019): F1649—F1655. http://dx.doi.org/10.1152/ajprenal.00395.2019.
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The severity of polycystic kidney diseases (PKD) depends on the counterbalancing of genetic predisposition and environmental factors exerting permissive or protective influence on cyst development. One poorly characterized phenomenon in the cystic epithelium is abnormal purinergic signaling. Earlier experimental studies revealed the high importance of the ionotropic P2X receptors (particularly, P2X7) in the pathophysiology of the cyst wall. To study mechanisms of P2X7 involvement in cyst growth and aspects of targeting these receptors in PKD treatment we performed a CRISPR/SpCas9-mediated global knockout of the P2rx7 gene in PCK rats, a model of autosomal recessive PKD (ARPKD). A single base insertion in exon 2 of the P2rx7 gene in the renal tissues of homozygous mutant animals leads to lack of P2X7 protein that did not affect their viability or renal excretory function. However, PCK. P2rx7 rats demonstrated slower cyst growth (but not formation of new cysts) compared with heterozygous and PCK. P2rx7+ littermates. P2X7 receptors are known to activate pannexin-1, a plasma channel capable of releasing ATP, and we found here that pannexin-1 expression in the cystic epithelium is significantly higher than in nondilated tubules. P2X7 deficiency reduces renal pannexin-1 protein expression and daily urinary ATP excretion. Patch-clamp analysis revealed that lack of P2X7 increases epithelial sodium channel activity in renal tissues and restores impaired channel activity in cysts. Interpretation of our current data in the context of earlier studies strongly suggests that P2X7 contributes to cyst growth by increasing pannexin-1-dependent pathogenic ATP release into the lumen and reduction of sodium reabsorption across the cyst walls.
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Inoue, Hiroyuki, Hidetaka Kuroda, Wataru Ofusa, Sadao Oyama, Maki Kimura, Tatsuya Ichinohe, and Yoshiyuki Shibukawa. "Functional Coupling between the P2X7 Receptor and Pannexin-1 Channel in Rat Trigeminal Ganglion Neurons." International Journal of Molecular Sciences 22, no. 11 (June 1, 2021): 5978. http://dx.doi.org/10.3390/ijms22115978.
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The ionotropic P2X receptor, P2X7, is believed to regulate and/or generate nociceptive pain, and pain in several neuropathological diseases. Although there is a known relationship between P2X7 receptor activity and pain sensing, its detailed functional properties in trigeminal ganglion (TG) neurons remains unclear. We examined the electrophysiological and pharmacological characteristics of the P2X7 receptor and its functional coupling with other P2X receptors and pannexin-1 (PANX1) channels in primary cultured rat TG neurons, using whole-cell patch-clamp recordings. Application of ATP and Bz-ATP induced long-lasting biphasic inward currents that were more sensitive to extracellular Bz-ATP than ATP, indicating that the current was carried by P2X7 receptors. While the biphasic current densities of the first and second components were increased by Bz-ATP in a concentration dependent manner; current duration was only affected in the second component. These currents were significantly inhibited by P2X7 receptor antagonists, while only the second component was inhibited by P2X1, 3, and 4 receptor antagonists, PANX1 channel inhibitors, and extracellular ATPase. Taken together, our data suggests that autocrine or paracrine signaling via the P2X7-PANX1-P2X receptor/channel complex may play important roles in several pain sensing pathways via long-lasting neuronal activity driven by extracellular high-concentration ATP following tissue damage in the orofacial area.
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Marques, Rita D., Pauline I. A. de Bruijn, Mads V. Sorensen, Markus Bleich, Helle A. Praetorius, and Jens Leipziger. "Basolateral P2X receptors mediate inhibition of NaCl transport in mouse medullary thick ascending limb (mTAL)." American Journal of Physiology-Renal Physiology 302, no. 4 (February 15, 2012): F487—F494. http://dx.doi.org/10.1152/ajprenal.00570.2011.
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Extracellular nucleotides regulate epithelial transport via luminal and basolateral P2 receptors. Renal epithelia express multiple P2 receptors, which mediate significant inhibition of solute absorption. Recently, we identified several P2 receptors in the medullary thick ascending limb (mTAL) including luminal and basolateral P2Y2 receptors (Jensen ME, Odgaard E, Christensen MH, Praetorius HA, Leipziger J. J Am Soc Nephrol 18: 2062–2070, 2007). In addition, we found evidence for a basolateral P2X receptor. Here, we investigate the effect of basolateral ATP on NaCl absorption in isolated, perfused mouse mTALs using the electrical measurement of equivalent short-circuit current ( I′sc). Nonstimulated mTALs transported at a rate of 1,197 ± 104 μA/cm2 ( n = 10), which was completely blockable with luminal furosemide (100 μM). Basolateral ATP (100 μM) acutely (1 min) and reversibly reduced the absorptive I′sc. After 2 min, the reduction amounted to 24.4 ± 4.0% ( n = 10). The nonselective P2 receptor antagonist suramin blocked the effect. P2Y receptors were found not to be involved in this effect. The P2X receptor agonist 2-methylthio ATP mimicked the ATP effect, and the P2X receptor antagonist periodate-oxidized ATP blocked it. In P2X7−/− mice, the ATP effect remained unaltered. In contrast, in P2X4−/− mice the ATP-induced inhibition of transport was reduced. A comprehensive molecular search identified P2X4, P2X5, and P2X1 receptor subunit mRNA in isolated mouse mTALs. These data define that basolateral ATP exerts a significant inhibition of Na+ absorption in mouse mTAL. Pharmacological, molecular, and knockout mouse data identify a role for the P2X4 receptor. We suggest that other P2X subunits like P2X5 are part of the P2X receptor complex. These data provide the novel perspective that an ionotropic receptor and thus a nonselective cation channel causes transport inhibition in an intact renal epithelium.
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Adamiak, Mateusz, Arjun Thapa, Kamila Bujko, Valentina Pensato, Magdalena Kucia, Janina Ratajczak, and Mariusz Z. Ratajczak. "A Novel Underappreciated Role for the Extracellular Adenosine Triphosphate (ATP)-P2X4 Purinergic Receptor Axis in the Homing and Engraftment of HSPCs." Blood 136, Supplement 1 (November 5, 2020): 32. http://dx.doi.org/10.1182/blood-2020-137123.
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Background. Adenosine triphosphate (ATP) is an important nucleotide involved in intracellular energy transfer, but when released from activated cells into the extracellular space as extracellular ATP (eATP) it becomes a crucial mediator of the purinergic signaling network. Purinergic receptors for extracellular nucleotides (EXNs), expressed on the surface of all cells in the body, are represented by the P1, P2X, and P2Y receptor families, which are among the most abundant receptors in living organisms. Of all these receptors, the P2X receptor family is most highly specific for eATP signaling and consists of seven members (P2X1-7). We found that human and murine hematopoietic stem progenitor cells (HSPCs) highly express two members of this family, the P2X4 and P2X7 receptors. We recently reported that both are involved in optimal mobilization of HSPCs by activating Nlrp3 inflammasome (Leukemia 2020 Jun;34(6):1512-1523 and Stem Cell Rev Rep. 2019 Jun;15(3):391-403). We also reported that the P2X7 receptor expressed on the surface of HSPCs facilitates the homing and engraftment of HSPCs by increasing their responsiveness to SDF-1 gradients. Interestingly, it has been proposed that both receptors heterodimerize to exert optimal activity. Hypothesis. Since, the P2X4 and P2X7 receptors show several similar biological effects in non-hematopoietic cells, we became interested in the role of the P2X4 receptor in homing and engraftment of HSPCs.Materials and Methods. To test this hypothesis, we isolated SKL cells from P2X4-KO mice and tested them for migration in response to BM chemoattractants, including the major homing factor SDF-1. Next, we tested the short- and long-term homing of mouse BM cells after exposure to the P2X4-specific inhibitor PBS12054 in normal mice by evaluating the number of donor-derived PKH67-labeled BMMNCs and CFU-GM clonogenic progenitors isolated from recipient mouse BM 24 hours after transplantation as well as the number of day-12 colony-forming units in spleen (CFU-S) and day-12 CFU-GM clonogenic progenitors. These data were confirmed in transplant studies employing P2X4-KO bone marrow cells. In parallel, we also evaluated the recovery kinetics of leukocytes and blood platelets in the PB of transplanted animals. Finally, we also perturbed P2X4 expression in transplanted mice with PBS12054 and studied the effect on homing and engraftment of normal BM cells, as described above. Results. We found that P2X4-KO mouse HSPCs have a defect in migration in response to BM chemoattractants involved in BM homing, including the major homing factor SDF-1 as well as the supportive factors S1P and eATP. Perturbation of P2X4 expression on the surface of HSPCs led to significant defective homing and engraftment of HSPCs. Moreover, inhibition of P2X4 in the recipient mouse BM microenvironment had a similar effect. Conclusions. We identified for the first time the role of eATP-P2X4 signaling in the homing and engraftment of HSPCs. To explain this result, we conclude that the eATP-P2X4 axis is, like the eATP-P2X7 axis, a potent activator of Nlrp3 inflammasomes and that defective eATP-P2X4 signaling impairs the role of purinergic signaling and the Nlrp3 inflammasome in homing and engraftment. Moreover, our results show a similar homing and engraftment phenotype for P2X4-KO mice as that seen in P2X7-KO animals, which provides functional support for the proposed dimerization of P2X7 with P2X4 receptors and the necessary presence of both receptors for optimal function. This question is currently being addressed in our laboratory by employing the fluorescence resonance energy transfer (FRET) technique. Finally, we provide additional evidence that, in addition to SDF-1 and S1P, eATP and purinergic signaling involving P2X4 and P2X7 receptors is an important and underappreciated regulator of HSPC trafficking and a potential target for molecular optimization of both processes. Disclosures No relevant conflicts of interest to declare.
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Stoop, Ron, Annmarie Surprenant, and R. Alan North. "Different Sensitivities to pH of ATP-Induced Currents at Four Cloned P2X Receptors." Journal of Neurophysiology 78, no. 4 (October 1, 1997): 1837–40. http://dx.doi.org/10.1152/jn.1997.78.4.1837.
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Stoop, Ron, Annmarie Surprenant, and R. Alan North. Different sensitivities to pH of ATP-induced currents at four cloned P2X receptors. J. Neurophysiol. 78: 1837–1840, 1997. The effect of changing extracellular pH was studied on the currents induced by ATP or αβ-methylene-ATP in HEK293 cells transfected with different P2X receptor subunits. In cells expressing P2X1, P2X3, or P2X4 receptors, the effect of ATP was decreased by acidification. In cells expressing P2X2 receptors, acidification increased the ATP-induced current; this effect was also seen in cells expressing heteromeric P2X2 and P2X3 receptors. At P2X2 receptors, acidification caused a leftward shift in the ATP concentration-response curve, without change in maximum; the pKa for this effect was 7.3. At P2X4 receptors, acidification caused a rightward shift in the ATP concentration-response curve, without change in the maximum; the pKa for this effect was 6.8. The pH dependence of the action of ATP should be taken into account in studies of synaptic transmission, and it may provide a further tool to assign molecular identity to P2X receptors expressed by brain neurons.
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Ziberi, Sihana, Mariachiara Zuccarini, Marzia Carluccio, Patricia Giuliani, Lucia Ricci-Vitiani, Roberto Pallini, Francesco Caciagli, Patrizia Di Iorio, and Renata Ciccarelli. "Upregulation of Epithelial-To-Mesenchymal Transition Markers and P2X7 Receptors Is Associated to Increased Invasiveness Caused by P2X7 Receptor Stimulation in Human Glioblastoma Stem Cells." Cells 9, no. 1 (December 29, 2019): 85. http://dx.doi.org/10.3390/cells9010085.
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Glioblastoma (GBM) stem cells (GSCs), which contribute to GBM unfavorable prognosis, show high expression levels of ATP/P2X7 receptors (P2X7R). Here, we reported that cells exposure to 2’(3’)-O-(4-benzoylbenzoyl)-ATP (BzATP), a P2X7R agonist, up-regulated the expression of markers associated to epithelial-to-mesenchymal transition (EMT), a process likely contributing to GSC malignancy, and increased GSC migration/invasiveness like the known EMT inducer, Transforming Growth Factor β1 (TGFβ1). These effects were coupled to phosphorylation of SMAD2, a downstream effector in the TGFβ pathway, suggesting its involvement in P2X7R-mediated activity in GSCs. All BzATP effects, including a decrease in the caspase 3/7 activity in GSC medium, were mostly counteracted by the P2X7R antagonist A438079. Finally, BzATP increased the subunit expression of two main human P2X7R splice variants, the full-length P2X7A and the truncated P2X7B, lacking the carboxylic tail, which have different functional properties depending on their arrangement. Since up-regulation of A/B subunits might favor their assembly into a heterotrimeric P2X7R with great sensitivity towards agonists and cell energy support, this is in line with increased EMT markers expression, cell migration/invasion and GSC survival observed following P2X7R stimulation. As in GBM microenvironment extracellular ATP levels may activate P2X7R, our data suggest a P2X7R role in GBM recurrence/invasiveness.
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Woehrle, Tobias, Linda Yip, Abdallah Elkhal, Yuka Sumi, Yu Chen, Yongli Yao, Paul A. Insel, and Wolfgang G. Junger. "Pannexin-1 hemichannel–mediated ATP release together with P2X1 and P2X4 receptors regulate T-cell activation at the immune synapse." Blood 116, no. 18 (November 4, 2010): 3475–84. http://dx.doi.org/10.1182/blood-2010-04-277707.
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Abstract Engagement of T cells with antigen-presenting cells requires T-cell receptor (TCR) stimulation at the immune synapse. We previously reported that TCR stimulation induces the release of cellular adenosine-5′-triphosphate (ATP) that regulates T-cell activation. Here we tested the roles of pannexin-1 hemichannels, which have been implicated in ATP release, and of various P2X receptors, which serve as ATP-gated Ca2+ channels, in events that control T-cell activation. TCR stimulation results in the translocation of P2X1 and P2X4 receptors and pannexin-1 hemichannels to the immune synapse, while P2X7 receptors remain uniformly distributed on the cell surface. Removal of extracellular ATP or inhibition, mutation, or silencing of P2X1 and P2X4 receptors inhibits Ca2+ entry, nuclear factors of activated T cells (NFAT) activation, and induction of interleukin-2 synthesis. Inhibition of pannexin-1 hemichannels suppresses TCR-induced ATP release, Ca2+ entry, and T-cell activation. We conclude that pannexin-1 hemichannels and P2X1 and P2X4 receptors facilitate ATP release and autocrine feedback mechanisms that control Ca2+ entry and T-cell activa-tion at the immune synapse.
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Thériault, Olivier, Hugo Poulin, George R. Thomas, Albert D. Friesen, Waleed A. Al-Shaqha, and Mohamed Chahine. "Pyridoxal-5′-phosphate (MC-1), a vitamin B6 derivative, inhibits expressed P2X receptors." Canadian Journal of Physiology and Pharmacology 92, no. 3 (March 2014): 189–96. http://dx.doi.org/10.1139/cjpp-2013-0404.
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P2X receptors are cation-permeable ligand-gated ion channels that open in response to the binding of ATP. These receptors are present in many excitable cells, including neurons, striated muscle cells, epithelial cells, and leukocytes. They mediate fast excitatory neurotransmission in the central and peripheral nervous systems and are thought to be involved in neuropathic pain, inflammation, and cell damage following ischemia–reperfusion injuries. P2X receptors are thus a target for the development of new therapeutics to treat chronic pain and inflammation. In this study, we characterized the inhibition caused by pyridoxal-5′-phosphate, a natural metabolite of vitamin B6 (MC-1), of P2X2, P2X4, P2X7, and P2X2/3 receptors stably expressed in HEK293 cells using the patch-clamp technique in the whole-cell configuration. We also tested a new approach using VC6.1, a modified cameleon calcium-sensitive fluorescent protein, to characterize the inhibition of P2X2 and P2X2/3. MC-1 blocked these two P2X receptors, with an IC50 of 7 and 13 μmol/L, respectively. P2X2 exhibited the highest affinity for VC6.1, and the chimeric receptor P2X2/3, the lowest. The patch-clamp and imaging approaches gave similar results and indicated that VC6.1 may be useful for high throughput drug screening. Pyridoxal-5′-phosphate is an efficient P2X blocker and can be classified as a P2X antagonist.
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Samways, Damien S. K., and Terrance M. Egan. "Acidic Amino Acids Impart Enhanced Ca2+ Permeability and Flux in Two Members of the ATP-gated P2X Receptor Family." Journal of General Physiology 129, no. 3 (February 26, 2007): 245–56. http://dx.doi.org/10.1085/jgp.200609677.
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P2X receptors are ATP-gated cation channels expressed in nerve, muscle, bone, glands, and the immune system. The seven family members display variable Ca2+ permeabilities that are amongst the highest of all ligand-gated channels (Egan and Khakh, 2004). We previously reported that polar residues regulate the Ca2+ permeability of the P2X2 receptor (Migita et al., 2001). Here, we test the hypothesis that the formal charge of acidic amino acids underlies the higher fractional Ca2+ currents (Pf%) of the rat and human P2X1 and P2X4 subtypes. We used patch-clamp photometry to measure the Pf% of HEK-293 cells transiently expressing a range of wild-type and genetically altered receptors. Lowering the pH of the extracellular solution reduced the higher Pf% of the P2X1 receptor but had no effect on the lower Pf% of the P2X2 receptor, suggesting that ionized side chains regulate the Ca2+ flux of some family members. Removing the fixed negative charges found at the extracellular ends of the transmembrane domains also reduced the higher Pf% of P2X1 and P2X4 receptors, and introducing these charges at homologous positions increased the lower Pf% of the P2X2 receptor. Taken together, the data suggest that COO− side chains provide an electrostatic force that interacts with Ca2+ in the mouth of the pore. Surprisingly, the glutamate residue that is partly responsible for the higher Pf% of the P2X1 and P2X4 receptors is conserved in the P2X3 receptor that has the lowest Pf% of all family members. We found that neutralizing an upstream His45 increased Pf% of the P2X3 channel, suggesting that this positive charge masks the facilitation of Ca2+ flux by the neighboring Glu46. The data support the hypothesis that formal charges near the extracellular ends of transmembrane domains contribute to the high Ca2+ permeability and flux of some P2X receptors.
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De Salis, Sophie K. F., Lanxin Li, Zheng Chen, Kam Wa Lam, Kristen K. Skarratt, Thomas Balle, and Stephen J. Fuller. "Alternatively Spliced Isoforms of the P2X7 Receptor: Structure, Function and Disease Associations." International Journal of Molecular Sciences 23, no. 15 (July 25, 2022): 8174. http://dx.doi.org/10.3390/ijms23158174.
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The P2X7 receptor (P2X7R) is an ATP-gated membrane ion channel that is expressed by multiple cell types. Following activation by extracellular ATP, the P2X7R mediates a broad range of cellular responses including cytokine and chemokine release, cell survival and differentiation, the activation of transcription factors, and apoptosis. The P2X7R is made up of three P2X7 subunits that contain specific domains essential for the receptor’s varied functions. Alternative splicing produces P2X7 isoforms that exclude one or more of these domains and assemble in combinations that alter P2X7R function. The modification of the structure and function of the P2X7R may adversely affect cellular responses to carcinogens and pathogens, and alternatively spliced (AS) P2X7 isoforms have been associated with several cancers. This review summarizes recent advances in understanding the structure and function of AS P2X7 isoforms and their associations with cancer and potential role in modulating the inflammatory response.
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North, R. Alan. "Molecular Physiology of P2X Receptors." Physiological Reviews 82, no. 4 (January 10, 2002): 1013–67. http://dx.doi.org/10.1152/physrev.00015.2002.
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P2X receptors are membrane ion channels that open in response to the binding of extracellular ATP. Seven genes in vertebrates encode P2X receptor subunits, which are 40–50% identical in amino acid sequence. Each subunit has two transmembrane domains, separated by an extracellular domain (∼280 amino acids). Channels form as multimers of several subunits. Homomeric P2X1, P2X2, P2X3, P2X4, P2X5, and P2X7channels and heteromeric P2X2/3and P2X1/5channels have been most fully characterized following heterologous expression. Some agonists (e.g., αβ-methylene ATP) and antagonists [e.g., 2′,3′- O-(2,4,6-trinitrophenyl)-ATP] are strongly selective for receptors containing P2X1and P2X3subunits. All P2X receptors are permeable to small monovalent cations; some have significant calcium or anion permeability. In many cells, activation of homomeric P2X7receptors induces a permeability increase to larger organic cations including some fluorescent dyes and also signals to the cytoskeleton; these changes probably involve additional interacting proteins. P2X receptors are abundantly distributed, and functional responses are seen in neurons, glia, epithelia, endothelia, bone, muscle, and hemopoietic tissues. The molecular composition of native receptors is becoming understood, and some cells express more than one type of P2X receptor. On smooth muscles, P2X receptors respond to ATP released from sympathetic motor nerves (e.g., in ejaculation). On sensory nerves, they are involved in the initiation of afferent signals in several viscera (e.g., bladder, intestine) and play a key role in sensing tissue-damaging and inflammatory stimuli. Paracrine roles for ATP signaling through P2X receptors are likely in neurohypophysis, ducted glands, airway epithelia, kidney, bone, and hemopoietic tissues. In the last case, P2X7receptor activation stimulates cytokine release by engaging intracellular signaling pathways.
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Liu, Cuiling, Simon Mather, Yu Huang, Christopher J. Garland, and Xiaoqiang Yao. "Extracellular ATP facilitates flow-induced vasodilatation in rat small mesenteric arteries." American Journal of Physiology-Heart and Circulatory Physiology 286, no. 5 (May 2004): H1688—H1695. http://dx.doi.org/10.1152/ajpheart.00576.2003.
Full textAbstract:
ATP can be released from endothelial cells, and this release is increased by intraluminal flow in blood vessels. In the present study, the effect of extracellular ATP (1 μM) on flow-induced vasodilatation was investigated in isolated and pressurized rat small mesenteric arteries. In the absence of extracellular ATP, only 46% of arteries developed dilatation in response to flow, and this response was both transient and unstable. In marked contrast, with ATP present, all vessels developed a prolonged and stable dilatation in response to flow. Even in the vessels that failed to respond to flow in the absence of ATP, dilatation could be stimulated once ATP was present. The ability of ATP to facilitate flow-induced vasodilatation was mimicked by UTP (1 μM), a P2Y agonist, or 3′- O-(4-benzoyl)benzoyl ATP (BzATP; 10 μM), an agonist for P2X1, P2X7, and P2Y11 purinoceptors. The involvement of P2X7 purinoceptors was further supported by the inhibitory effect of KN-62 (1 μM), a P2X7 antagonist, on the action of BzATP. P2X1 and P2X3 purinoceptors were not involved because their receptor agonist α,β-methylene ATP had no effect. The facilitating effect of ATP on flow dilatation was also attenuated by the combined application of reactive blue 2 (100 μM), a P2Y antagonist, and suramin (100 μM), a nonselective P2X and P2Y antagonist. Furthermore, flow-induced dilatation obtained in the presence of ATP was reproducible. In contrast, in the additional presence of the ectonucleotidase inhibitor ARL-67156 (10 μM), although the first dilatation was normal, the responses to the second and later exposures to flow were greatly attenuated. The nonhydrolyzable ATP analogs adenosine-5′-(3-thiotriphosphate)trilithium salt (1 μM) and adenosine 5′-(β,γ-imido) triphosphate tetralithium salt hydrate (10 μM) had similar effects to those of ARL-67156. These data suggest that ATP acts through both P2X and P2Y purinoceptors to facilitate flow-induced vasodilatation and that ectonucleotidases prevent this effect by degrading ATP on the endothelial cell surface.
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Panupinthu, Nattapon, Joseph T. Rogers, Lin Zhao, Luis Pastor Solano-Flores, Fred Possmayer, Stephen M. Sims, and S. Jeffrey Dixon. "P2X7 receptors on osteoblasts couple to production of lysophosphatidic acid: a signaling axis promoting osteogenesis." Journal of Cell Biology 181, no. 5 (June 2, 2008): 859–71. http://dx.doi.org/10.1083/jcb.200708037.
Full textAbstract:
Nucleotides are released from cells in response to mechanical stimuli and signal in an autocrine/paracrine manner through cell surface P2 receptors. P2rx7−/− mice exhibit diminished appositional growth of long bones and impaired responses to mechanical loading. We find that calvarial sutures are wider in P2rx7−/− mice. Functional P2X7 receptors are expressed on osteoblasts in situ and in vitro. Activation of P2X7 receptors by exogenous nucleotides stimulates expression of osteoblast markers and enhances mineralization in cultures of rat calvarial cells. Moreover, osteogenesis is suppressed in calvarial cell cultures from P2rx7−/− mice compared with the wild type. P2X7 receptors couple to production of the potent lipid mediators lysophosphatidic acid (LPA) and prostaglandin E2. Either an LPA receptor antagonist or cyclooxygenase (COX) inhibitors abolish the stimulatory effects of P2X7 receptor activation on osteogenesis. We conclude that P2X7 receptors enhance osteoblast function through a cell-autonomous mechanism. Furthermore, a novel signaling axis links P2X7 receptors to production of LPA and COX metabolites, which in turn stimulate osteogenesis.
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Agrawal, Ankita, and Alison Gartland. "P2X7 receptors: role in bone cell formation and function." Journal of Molecular Endocrinology 54, no. 2 (January 14, 2015): R75—R88. http://dx.doi.org/10.1530/jme-14-0226.
Full textAbstract:
The role of the P2X7 receptor (P2X7R) is being explored with intensive interest in the context of normal bone physiology, bone-related diseases and, to an extent, bone cancer. In this review, we cover the current understanding of P2X7R regulation of bone cell formation, function and survival. We will discuss how the P2X7R drives lineage commitment of undifferentiated bone cell progenitors, the vital role of P2X7R activation in bone mineralisation and its relatively unexplored role in osteocyte function. We also review how P2X7R activation is imperative for osteoclast formation and its role in bone resorption via orchestrating osteoclast apoptosis. Variations in the gene for the P2X7R (P2RX7) have implications for P2X7R-mediated processes and we review the relevance of these genetic variations in bone physiology. Finally, we highlight how targeting P2X7R may have therapeutic potential in bone disease and cancer.
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Mikolajewicz, Nicholas, Delaney Smith, Svetlana V. Komarova, and Anmar Khadra. "High-affinity P2Y2 and low-affinity P2X7 receptor interaction modulates ATP-mediated calcium signaling in murine osteoblasts." PLOS Computational Biology 17, no. 6 (June 21, 2021): e1008872. http://dx.doi.org/10.1371/journal.pcbi.1008872.
Full textAbstract:
The P2 purinergic receptor family implicated in many physiological processes, including neurotransmission, mechanical adaptation and inflammation, consists of ATP-gated non-specific cation channels P2XRs and G-protein coupled receptors P2YRs. Different cells, including bone forming osteoblasts, express multiple P2 receptors; however, how P2X and P2Y receptors interact in generating cellular responses to various doses of [ATP] remains poorly understood. Using primary bone marrow and compact bone derived osteoblasts and BMP2-expressing C2C12 osteoblastic cells, we demonstrated conserved features in the P2-mediated Ca2+ responses to ATP, including a transition of Ca2+ response signatures from transient at low [ATP] to oscillatory at moderate [ATP], and back to transient at high [ATP], and a non-monotonic changes in the response magnitudes which exhibited two troughs at 10−4 and 10−2 M [ATP]. We identified P2Y2 and P2X7 receptors as predominantly contributing to these responses and constructed a mathematical model of P2Y2R-induced inositol trisphosphate (IP3) mediated Ca2+ release coupled to a Markov model of P2X7R dynamics to study this system. Model predictions were validated using parental and CRISPR/Cas9-generated P2Y2 and P2Y7 knockouts in osteoblastic C2C12-BMP cells. Activation of P2Y2 by progressively increasing [ATP] induced a transition from transient to oscillatory to transient Ca2+ responses due to the biphasic nature of IP3Rs and the interaction of SERCA pumps with IP3Rs. At high [ATP], activation of P2X7R modulated the response magnitudes through an interplay between the biphasic nature of IP3Rs and the desensitization kinetics of P2X7Rs. Moreover, we found that P2Y2 activity may alter the kinetics of P2X7 towards favouring naïve state activation. Finally, we demonstrated the functional consequences of lacking P2Y2 or P2X7 in osteoblast mechanotransduction. This study thus provides important insights into the biophysical mechanisms underlying ATP-dependent Ca2+ response signatures, which are important in mediating bone mechanoadaptation.
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Spildrejorde, Mari, Rachael Bartlett, Leanne Stokes, Iman Jalilian, Michelle Peranec, Vanessa Sluyter, Belinda L. Curtis, et al. "R270C polymorphism leads to loss of function of the canine P2X7 receptor." Physiological Genomics 46, no. 14 (July 15, 2014): 512–22. http://dx.doi.org/10.1152/physiolgenomics.00195.2013.
Full textAbstract:
The relative function of the P2X7 receptor, an ATP-gated ion channel, varies between humans due to polymorphisms in the P2RX7 gene. This study aimed to assess the functional impact of P2X7 variation in a random sample of the canine population. Blood and genomic DNA were obtained from 69 dogs selected as representatives of a cross section of different breeds. P2X7 function was determined by flow cytometric measurements of dye uptake and patch-clamp measurements of inward currents. P2X7 expression was determined by immunoblotting and immunocytochemistry. Sequencing was used to identify P2RX7 gene polymorphisms. P2X7 was cloned from an English springer spaniel, and point mutations were introduced into this receptor by site-directed mutagenesis. The relative function of P2X7 on monocytes varied between individual dogs. The canine P2RX7 gene encoded four missense polymorphisms: F103L and P452S, found in heterozygous and homozygous dosage, and R270C and R365Q, found only in heterozygous dosage. Moreover, R270C and R365Q were associated with the cocker spaniel and Labrador retriever, respectively. F103L, R270C, and R365Q but not P452S corresponded to decreased P2X7 function in monocytes but did not explain the majority of differences in P2X7 function between dogs, indicating that other factors contribute to this variability. Heterologous expression of site-directed mutants of P2X7 in human embryonic kidney-293 cells indicated that the R270C mutant was nonfunctional, the F103L and R365Q mutants had partly reduced function, and the P452S mutant functioned normally. Taken together, these data highlight that a R270C polymorphism has major functional impact on canine P2X7.
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Hasko, George, Balazs Csoka, Zoltan Németh, Gabor Törö, Marco Idzko, and Andreas Zech. "ATP protects against sepsis through macrophage P2X7 purinergic receptors by enhancing bacterial killing (INC2P.407)." Journal of Immunology 194, no. 1_Supplement (May 1, 2015): 55.1. http://dx.doi.org/10.4049/jimmunol.194.supp.55.1.
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Abstract Extracellular ATP binds to and signals through P2X7 receptors (P2X7R)s to modulate immune function in both inflammasome-dependent and independent manners. We show here using P2X7-/- mice as well as pharmacological receptor and channel ligands that ATP release through connexin/pannexin channels and subsequent P2X7R activation are crucial for the control of mortality, bacterial dissemination and inflammation following sepsis induced by cecal ligation and puncture. Our results with P2X7-/- bone-marrow chimeras, adoptive transfer of macrophages, and myeloid-specific P2X7-/- mice indicate that P2X7R signaling on macrophages is required for the protective effect of P2X7Rs. P2X7R signaling protects through enhancing bacterial killing by macrophages, but independently of the inflammasome. In summary, targeting P2X7Rs provides a new opportunity for harnessing an endogenous protective immune mechanism in the therapy of sepsis.
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LIGHT, ALAN R., YING WU, RONALD W. HUGHEN, and PETER B. GUTHRIE. "Purinergic receptors activating rapid intracellular Ca2+ increases in microglia." Neuron Glia Biology 2, no. 2 (December 1, 2005): 125–38. http://dx.doi.org/10.1017/s1740925x05000323.
Full textAbstract:
We provide both molecular and pharmacological evidence that the metabotropic, purinergic, P2Y6, P2Y12 and P2Y13 receptors and the ionotropic P2X4 receptor contribute strongly to the rapid calcium response caused by ATP and its analogues in mouse microglia. Real-time PCR demonstrates that the most prevalent P2 receptor in microglia is P2Y6 followed, in order, by P2X4, P2Y12, and P2X7 = P2Y13. Only very small quantities of mRNA for P2Y1, P2Y2, P2Y4, P2Y14, P2X3 and P2X5 were found. Dose-response curves of the rapid calcium response gave a potency order of: 2MeSADP>ADP=UDP=IDP=UTP>ATP>BzATP, whereas A2P4 had little effect. Pertussis toxin partially blocked responses to 2MeSADP, ADP and UDP. The P2X4 antagonist suramin, but not PPADS, significantly blocked responses to ATP. These data indicate that P2Y6, P2Y12, P2Y13 and P2X receptors mediate much of the rapid calcium responses and shape changes in microglia to low concentrations of ATP, presumably at least partly because ATP is rapidly hydrolyzed to ADP. Expression of P2Y6, P2Y12 and P2Y13 receptors appears to be largely glial in the brain, so that peripheral immune cells and CNS microglia share these receptors. Thus, purinergic, metabotropic, P2Y6, P2Y12, P2Y13 and P2X4 receptors might share a role in the activation and recruitment of microglia in the brain and spinal cord by widely varying stimuli that cause the release of ATP, including infection, injury and degeneration in the CNS, and peripheral tissue injury and inflammation which is signaled via nerve signaling to the spinal cord.
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Pacheco, Paulo Anastácio Furtado, Daniel Tadeu Gomes Gonzaga, Natalia Lidmar von Ranke, Carlos Rangel Rodrigues, David Rodrigues da Rocha, Fernando de Carvalho da Silva, Vitor Francisco Ferreira, and Robson Xavier Faria. "Synthesis, Biological Evaluation and Molecular Modeling Studies of Naphthoquinone Sulfonamides and Sulfonate Ester Derivatives as P2X7 Inhibitors." Molecules 28, no. 2 (January 6, 2023): 590. http://dx.doi.org/10.3390/molecules28020590.
Full textAbstract:
ATP acts in the extracellular environment as an important signal, activating a family of receptors called purinergic receptors. In recent years, interest in the potential therapeutics of purinergic components, including agonists and antagonists of receptors, has increased. Currently, many observations have indicated that ATP acts as an important mediator of inflammatory responses and, when found in high concentrations in the extracellular space, is related to the activation of the P2X7 purinergic receptor. In this sense, the search for new inhibitors for this receptor has attracted a great deal of attention in recent years. Sulfonamide derivatives have been reported to be potent inhibitors of P2X receptors. In this study, ten naphthoquinone sulfonamide derivatives and five naphthoquinone sulfonate ester derivatives were tested for their inhibitory activity on the P2X7 receptor expressed in peritoneal macrophages. Some compounds showed promising results, displaying IC50 values lower than that of A740003. Molecular docking and dynamic studies also indicated that the active compounds bind to an allosteric site on P2X7R. The binding free energy indicates that sulfonamides have an affinity for the P2X7 receptor similar to A740003. Therefore, the compounds studied herein present potential P2X7R inhibition.
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Salvestrini, Valentina, Stefania Orecchioni, Francesca Reggiani, Giovanna Talarico, Elisa Orioli, Elena Adinolfi, Francesco Bertolini, et al. "P2X7 Receptor Activation By ATP As Target of Novel Therapies in Acute Myeloid Leukemia." Blood 126, no. 23 (December 3, 2015): 3684. http://dx.doi.org/10.1182/blood.v126.23.3684.3684.
Full textAbstract:
Abstract ATP is the key energy molecule as well as an ubiquitous extracellular messenger. Depending on its dose and the engaged purinergic P2 receptor (P2R) subtype, ATP can trigger many different cell responses, including proliferation and cell death. Recent studies have shown that high ATP level exhibits direct cytotoxicity on many tumor cell types. Among the receptors engaged by ATP, P2X7 is the most consistently expressed by tumor cells and its overexpression is related to tumor growth and progression. The P2X7 is an ATP-gated ion channel that, upon sustained stimulation with millimolar ATP concentrations, drives the opening of a non-selective large conductance pore, triggering cell-death signal. We previously demonstrated that ATP is a potent stimulator of normal hematopoietic stem cell compartment while inhibiting acute myeloid leukemia (AML) cells. Based on this observation, we studied AML samples (n=20) collected from the bone marrow or the peripheral blood of leukemic patients at diagnosis before treatment (percentage of circulating blasts >90%). In addition, normal hematopoietic stem cells (HSC) were isolated from leukapheresis products of 5 healthy donors receiving G-CSF. Our data demonstrate that AML cells express high level of P2X7 and that its activation with high dose of ATP reduces blast cell viability while is not effective on normal CD34+ cells. The cytotoxic effect is due to the induction of apoptosis, associated with reduction of mithocondrial membrane potential and activation of caspase cascade. Interestingly, P2X7 is also expressed by leukemic stem/progenitor cells (LSC) and ATP treatment exerts a direct cytotoxicity on different subsets of stem/progenitor cell compartment i.e. CD34- CD38-, CD34+ CD38-, CD34+ CD38+ and CD34- CD38+. Of note, this cytotoxic effect was not observed on HSC subpopulations. Furthermore, we transplanted 1x106 human AML cells into NSG immunodeficient mice followed by intraperitoneal administration of ATP every other day for thirty days post-transplantation. Our results show a 40% inhibition of AML engraftment in ATP-treated mice vs controls. Different P2X7 splice variants have been identified among which only two are functional: P2X7A, which shows both pro-apoptotic and trophic activity and P2X7B, which retains only the growth promoting phenotype. In order to explain ATP different effects on LSCs and normal HSCs, we assumed a different P2X7 isoforms expression on normal and leukemic cells. Preliminary results showed a reduced expression of both P2X7A and P2X7B on normal CD34+ compared to leukemic cells. In particular normal CD34+ express very low level of P2X7A, which is responsible for pore formation after ATP stimulation. Moreover, since P2X7 pore formation facilitates the passage of hydrophilic chemotherapeutic agents, we hypothesized that ATP may potentiate the cytotoxic effect of antineoplastic drugs. Our results showed that ATP potentiates the cytotoxic effect of ARA-C, by significantly reducing cell proliferation and increasing apoptosis of leukemia cell lines. In conclusion, overall survival of adult AML remains poor due to the lack of novel and effective therapies. Novel compounds that have the potential to improve the treatment efficacy with low toxicity are highly warranted. Overall, our results may provide the biological rationale to use P2X7 as a target for novel therapeutical approaches against AML. Disclosures Cavo: Janssen-Cilag, Celgene, Amgen, BMS: Honoraria.
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Estrada, Juan A., Guillaume P. Ducrocq, Joyce S. Kim, and Marc P. Kaufman. "Intrathecal injection of brilliant blue G, a P2X7 antagonist, attenuates the exercise pressor reflex in rats." American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 319, no. 2 (August 1, 2020): R223—R232. http://dx.doi.org/10.1152/ajpregu.00093.2020.
Full textAbstract:
Purinergic 2X (P2X) receptors on the endings of group III and IV afferents play a role in evoking the exercise pressor reflex. Particular attention has been paid to P2X3 receptors because their blockade in the periphery attenuated this reflex. In contrast, nothing is known about the role played by P2X receptors in the spinal cord in evoking the exercise pressor reflex in rats. P2X7 receptors, in particular, may be especially important in this regard because they are found in abundance on spinal glial cells and may communicate with neurons to effect reflexes controlling cardiovascular function. Consequently, we investigated the role played by spinal P2X7 receptors in evoking the exercise pressor reflex in decerebrated rats. We found that intrathecal injection of the P2X7 antagonist brilliant blue G (BBG) attenuated the exercise pressor reflex (blood pressure index: 294 ± 112 mmHg·s before vs. 7 ± 32 mmHg·s after; P < 0.05). Likewise, intrathecal injection of minocycline, which inhibits microglial cell output, attenuated the reflex. In contrast, intrathecal injection of BBG did not attenuate the pressor response evoked by intracarotid injection of sodium cyanide, a maneuver that stimulated carotid chemoreceptors. Moreover, injections of BBG either into the arterial supply of the contracting hindlimb muscles or into the jugular vein did not attenuate the exercise pressor reflex. Our findings support the hypothesis that P2X7 receptors on microglial cells within the spinal cord play a role in evoking the exercise pressor reflex.
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Faccinetto, Celine, Christelle Lecut, Roland Greimers, Vincent Bours, and Cecile Oury. "New Role for ATP P2X1 Receptors in the Control of Neutrophil Apoptosis and Respiratory Burst Activity." Blood 108, no. 11 (November 16, 2006): 1647. http://dx.doi.org/10.1182/blood.v108.11.1647.1647.
Full textAbstract:
Abstract P2X receptors are membrane non-selective cation channels that open in response to the binding of extracellular ATP. Seven genes encode P2X receptor subunits (P2X1–7) in vertebrates. Channels form homo- or hetero-multimers of several subunits that are highly permeable to Ca2+. P2X receptors are widely distributed. In recent years, increasing attention has been paid to extracellular ATP as a candidate danger signal locally released at the inception of inflammation. One of the most striking features of ATP is its ability to promote P2X7 receptor-mediated massive release of mature IL-1β from LPS-primed mononuclear phagocytes. On neutrophils, ATP rises intracellular Ca2+ concentrations, contributes to degranulation, adhesion, oxidative burst and delays apoptosis, events that may partly depend on the G-protein coupled P2Y2 receptor and on P2X7. In the present work, RT-PCR, Western blotting and immunofluorescence experiments reveal that neutrophils also express P2X1 and P2X5 receptor subtypes. A microarray analysis indicates that a 3-hour treatment of human peripheral blood neutrophils with the selective P2X1 and P2X1/5 receptor agonist, a,β-meATP, changes the expression of genes mainly involved in the control of cell fate. Accordingly, this agonist causes an increase of phosphatidylserine exposure on neutrophil membranes, maximally occurring after 3 hours and lasting until 18 hours of culture. In the presence of the protein synthesis inhibitor cycloheximide, a,β -meATP promotes caspase-3-dependent neutrophil apoptosis after 3 hours, which is correlated with highly reduced Fcγ RIII (CD16) membrane expression. In addition, a 1 min pretreatment of neutrophils with a,β -meATP potently increases tumor necrosis factor-a (TNF-α )-driven priming (30 min) of the respiratory burst induced by the bacterially derived peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP). Furthermore, a,β -meATP produces L-selectin (CD62L) shedding by its own and in an additive manner with TNF-α . This agonist also induces rapid and reversible phosphorylation of the survival kinases ERK (starting after 2 min) and Akt (15 min) as well as phosphorylation and degradation (after 2 min) of I-κ Bα , an inhibitor of the anti-apoptotic transcription factor NF-κ B. Hence, neutrophil P2X1 receptors might have a dual role in inflammation; they would both contribute to neutrophil activation and promote cell death of neutrophils that have reached the end of their useful life span. Activation of P2X1 receptors by extracellular ATP may thus represent novel regulatory mechanisms that govern neutrophil function.
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Harris, T. H., M. R. Wallace, H. Huang, H. Li, and L. M. Shaddox. "Associations of P2RX7 Functional Diplotypes with Localized Aggressive Periodontitis." JDR Clinical & Translational Research 4, no. 4 (July 18, 2019): 342–51. http://dx.doi.org/10.1177/2380084419863789.
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Aim: The purpose of this study was to test for the role of the P2X7 receptor in localized aggressive periodontitis (LAP). Methods: Peripheral blood was obtained from 95 subjects with LAP and 76 healthy unrelated controls (HUCs). Three P2RX7 single-nucleotide polymorphisms (rs1718119, rs2230911, and rs3751143) were genotyped from these subjects, and their peripheral blood samples were stimulated with lipopolysaccharide (LPS) from Escherichia coli and tested for inflammatory markers. The 3 P2RX7 single-nucleotide polymorphisms were in found to be in perfect linkage disequilibrium, and a total of 4 haplotypes and 9 diplotypes were identified among all subjects. For both subject populations, the 9 diplotypes were grouped into 4 functional groups and tested for association with subject inflammatory response. To specifically study the effects of extrinsic activation of the P2X7 receptor in LAP, peripheral blood samples from were stimulated under 3 treatments: LPS, LPS + ATP, and LPS +ATP+ P2X7 selective inhibitor. The effects of these treatments on P2X7 receptor activity were measured through Luminex protein assay. Last, to test whether receptor stimulation was related to P2RX7 expression, relative mRNA levels of P2RX7 were quantified with real-time quantitative polymerase chain reaction. Results: Several associations between the P2RX7 diplotypes and LPS-stimulated blood chemokine/cytokine levels were found between the LAP and HUC populations (P < 0.05). P2X7 activation resulted in statistically significant differences in IL-1β and IL-12p40 concentrations for both subject populations. The relative P2RX7 mRNA levels increased significantly after addition of its inhibitor for both LAP and HUC populations. Conclusions: This study detected an association between P2RX7 functional diplotypes and in vitro immune response of whole blood from subjects with LAP. In addition, we found that inhibition of the activated P2X7 receptor leads to increased P2RX7 mRNA levels, suggesting a feedback loop ( ClinicalTrials.gov NCT01330719). Knowledge Transfer Statement: The results of this study suggest that P2RX7 functional diplotypes are associated with LAP and their in vitro immune response to bacteria. Ongoing studies to uncover the mechanistic link between P2RX7 and LAP phenotypes could lead to the development of preventive approaches for susceptible subjects.
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Wu, Ping, Guohua Zhou, Xiaoqi Wu, Run Lv, Jiaqi Yao, and Qingping Wen. "P2X7 receptor induces microglia polarization to the M1 phenotype in cancer-induced bone pain rat models." Molecular Pain 18 (January 2022): 174480692110609. http://dx.doi.org/10.1177/17448069211060962.
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Background The transition from pro-inflammatory M1 phenotype to anti-inflammatory M2 phenotype presents a novel therapeutic strategy for chronic pain. Objective We investigated the role of microglia polarization in cancer-induced bone pain (CIBP), as well as the role of the P2X7 receptor in modulating M1 to M2 polarization. Methods Walker-256 breast cancer cells were administered into tibias of female rats to induce bone cancer–associated cancer. Results During bone cancer development, the P2X7 receptor and M1 microglia markers were upregulated. In contrast, inhibition of the P2X7 receptor by BBG, a blood-brain barrier-permeable P2X7R-specific antagonist, alleviated the pain and promoted microglia polarization toward the M2 phenotype, while suppressing the M1 phenotype in vivo and in vitro. Conclusion P2X7 receptor-mediated spinal microglia polarization is involved in alleviation of CIBP. Therefore, P2X7R is a potential option for CIBP treatment.
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He, Jin-Rong, Shu-Guang Yu, Yong Tang, and Peter Illes. "Purinergic signaling as a basis of acupuncture-induced analgesia." Purinergic Signalling 16, no. 3 (June 23, 2020): 297–304. http://dx.doi.org/10.1007/s11302-020-09708-z.
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Abstract This review summarizes experimental evidence indicating that purinergic mechanisms are causally involved in acupuncture (AP)-induced analgesia. Electroacupuncture (EAP) and manual AP release at pain-relevant acupoints ATP which may activate purinergic P2X receptors (Rs) especially of the P2X3 type situated at local sensory nerve endings (peripheral terminals of dorsal root ganglion [DRG] neurons); the central processes of these neurons are thought to inhibit via collaterals of ascending dorsal horn spinal cord neurons, pain-relevant pathways projecting to higher centers of the brain. In addition, during AP/EAP non-neuronal P2X4 and/or P2X7Rs localized at microglial cells of the CNS become activated at the spinal or supraspinal levels. In consequence, these microglia secrete bioactive compounds such as growth factors, cytokines, chemokines, reactive oxygen, and nitrogen species, which modulate the ascending neuronal pathways conducting painful stimuli. Alternatively, ATP released at acupoints by AP/EAP may be enzymatically degraded to adenosine, stimulating in loco presynaptic A1Rs exerting an inhibitory influence on the primary afferent fibers (the above mentioned pain-sensing peripheral terminals of DRG neurons) which thereby fail to conduct action potentials to the spinal cord dorsal horn. The net effect of the stimulation of P2X3, P2X4, P2X7, and A1Rs by the AP/EAP-induced release of ATP/adenosine at certain acupoints will be analgesia.
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Le Feuvre, Rosalind A., David Brough, Omar Touzani, and Nancy J. Rothwell. "Role of P2X7 Receptors in Ischemic and Excitotoxic Brain Injury In Vivo." Journal of Cerebral Blood Flow & Metabolism 23, no. 3 (March 2003): 381–84. http://dx.doi.org/10.1097/01.wcb.0000048519.34839.97.
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Purinergic P2X7 receptors may affect neuronal cell death through their ability to regulate the processing and release of interleukin-1β (IL-1β), a key mediator in neurodegeneration. The authors tested the hypothesis that ATP, acting at P2X7 receptors, contributes to experimentally induced neuronal death in rodents in vivo. Deletion of P2X7 receptors (P2X7 knockout mice) did not affect cell death induced by temporary cerebral ischemia, which was reduced by treatment with IL-1 receptor antagonist (IL-1RA). Treatment of mice with P2X antagonists did not affect ischemic or excitotoxic cell death, suggesting that P2X7 receptors are not primary mediators of experimentally induced neuronal death.
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Morelli, Anna, Paola Chiozzi, Anna Chiesa, Davide Ferrari, Juana M. Sanz, Simonetta Falzoni, Paolo Pinton, Rosario Rizzuto, Michael F. Olson, and Francesco Di Virgilio. "Extracellular ATP Causes ROCK I-dependent Bleb Formation in P2X7-transfected HEK293 Cells." Molecular Biology of the Cell 14, no. 7 (July 2003): 2655–64. http://dx.doi.org/10.1091/mbc.02-04-0061.
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The P2X7 ATP receptor mediates the cytotoxic effect of extracellular ATP. P2X7-dependent cell death is heralded by dramatic plasma membrane bleb formation. Membrane blebbing is a complex phenomenon involving as yet poorly characterized intracellular pathways. We have investigated the effect of extracellular ATP on HEK293 cells transfected with the cytotoxic/pore-forming P2X7 receptor. Addition of ATP to P2X7-transfected, but not to wt P2X7-less, HEK293 cells caused massive membrane blebbing within 1–2 min. UTP, a nucleotide incapable of activating P2X7, had no early effects on cell shape and bleb formation. Bleb formation triggered by ATP was reversible and required extracellular Ca2+ and an intact cytoskeleton. Furthermore, it was completely prevented by preincubation with the P2X blocker oxidized ATP. It was recently observed that the ROCK protein is a key determinant of bleb formation. Preincubation of HEK293-P2X7 cells with the ROCK blocker Y-27632 completely prevented P2X7-dependent blebbing. Although ATP triggered cleavage of the ROCK I isoform in P2X7-transfected HEK293 cells, the wide range caspase inhibitor z-VAD-fluoromethylketone had no effect. These observations suggest that P2X7-dependent plasma membrane blebbing depends on the activation of the serine/threonine kinase ROCK I.
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Solini, Anna, Sabina Cuccato, Davide Ferrari, Eleonora Santini, Sara Gulinelli, Maria Giulia Callegari, Angela Dardano, et al. "Increased P2X7 Receptor Expression and Function in Thyroid Papillary Cancer: A New Potential Marker of the Disease?" Endocrinology 149, no. 1 (October 18, 2007): 389–96. http://dx.doi.org/10.1210/en.2007-1223.
Full textAbstract:
Nucleotides are increasingly recognized as nonredundant extracellular signals for chemotaxis, cell growth, and cytokine release. Effects of extracellular nucleotides are mediated by P2 receptors, among which the P2X7 subtype is attracting increasing attention for its involvement in apoptosis, cell growth, and cytokine release. Recent studies showed that P2X7 is overexpressed in chronic lymphocytic leukemia and breast and prostate cancer. The aim of the present study was to better understand the clinical significance of P2X7 receptor expression in normal and cancer human thyroid tissues. P2X7 receptor message and protein expression and functional activity were tested in two cell lines (FB1 and FB2) established from either anaplastic or papillary primary thyroid cancer and in several histological samples of human papillary cancer. We show here that human thyroid papillary carcinoma, whether of the classical or follicular variant, expresses the P2X7 receptor (P2X7R) to a much higher level than normal thyroid tissue. The P2X7R was similarly up-regulated in FB1 and FB2 cell lines. In contrast to normal thyroid cells, both cell lines responded to extracellular nucleotide stimulation with a large increase in intracellular Ca2+ and secretion of IL-6. Ca2+ increase was attenuated and release of IL-6 was fully blocked by P2X7R inhibitors. Finally, the thyroid carcinoma cell lines had at least a 3-fold higher intracellular ATP concentration and maintained at least a 3-fold higher extracellular ATP level, compared with control cells. These data suggest that an enhanced P2X7R function might be a feature of human thyroid cancer.
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Cuthbertson, Peter, Amal Elhage, Dena Al-Rifai, Reece A. Sophocleous, Ross J. Turner, Ashraf Aboelela, Hiwa Majed, et al. "6-Furopyridine Hexamethylene Amiloride Is a Non-Selective P2X7 Receptor Antagonist." Biomolecules 12, no. 9 (September 16, 2022): 1309. http://dx.doi.org/10.3390/biom12091309.
Full textAbstract:
P2X7 is an extracellular adenosine 5′-triphopshate (ATP)-gated cation channel present on leukocytes, where its activation induces pro-inflammatory cytokine release and ectodomain shedding of cell surface molecules. Human P2X7 can be partially inhibited by amiloride and its derivatives at micromolar concentrations. This study aimed to screen a library of compounds derived from amiloride or its derivative 5-(N,N-hexamethylene) amiloride (HMA) to identify a potential P2X7 antagonist. 6-Furopyridine HMA (6-FPHMA) was identified as a novel P2X7 antagonist and was characterized further. 6-FPHMA impaired ATP-induced dye uptake into human RPMI8226 multiple myeloma cells and human P2X7-HEK293 cells, in a concentration-dependent, non-competitive manner. Likewise, 6-FPHMA blocked ATP-induced Ca2+ fluxes in human P2X7-HEK293 cells in a concentration-dependent, non-competitive manner. 6-FPHMA inhibited ATP-induced dye uptake into human T cells, and interleukin-1β release within human blood and CD23 shedding from RPMI8226 cells. 6-FPHMA also impaired ATP-induced dye uptake into murine P2X7- and canine P2X7-HEK293 cells. However, 6-FPHMA impaired ATP-induced Ca2+ fluxes in human P2X4-HEK293 cells and non-transfected HEK293 cells, which express native P2Y1, P2Y2 and P2Y4. In conclusion, 6-FPHMA inhibits P2X7 from multiple species. Its poor selectivity excludes its use as a specific P2X7 antagonist, but further study of amiloride derivatives as P2 receptor antagonists is warranted.
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Hashimoto-Hill, Seika, Leon Friesen, Myunghoo Kim, and Chang H. Kim. "Contraction of intestinal effector T cells by retinoic acid-induced purinergic receptor P2X7." Journal of Immunology 198, no. 1_Supplement (May 1, 2017): 65.15. http://dx.doi.org/10.4049/jimmunol.198.supp.65.15.
Full textAbstract:
Abstract Vitamin A is a key regulator of immunity and immune tolerance. Here we report that the vitamin A metabolite retinoic acid regulates the population size of intestinal effector T cells by inducing purinergic receptor P2X7 expression. RA induces P2X7 expression by direct binding to an intragenic enhancer of the P2rx7 gene in CD4+T cells, rendering them highly susceptible to apoptosis triggered by P2X7 activation in response to extracellular ATP and NAD. Utilizing P2X7-null mice, we found that this tolerogenic mechanism is critical for reining in the expansion of Th1 and Th17 effector T cell populations in the intestine where the concentrations of extracellular ATP/NAD and retinoic acid are high. We demonstrated that P2X7 activation can ameliorate colitis by inducing the apoptosis of inflammatory effector T cells. We conclude that the RA-P2X7 axis is a novel immune homeostatic mechanism by which the size of potentially inflammatory effector T cell populations is tightly controlled to prevent inflammatory diseases.