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1
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.
2
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.
3
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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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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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.
6
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.
7
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.
8
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.
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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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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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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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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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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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Nunes, Ana R., Raul Chavez-Valdez, Tarrah Ezell, David F. Donnelly, Joel C. Glover, and Estelle B. Gauda. "Effect of development on [Ca2+]i transients to ATP in petrosal ganglion neurons: a pharmacological approach using optical recording." Journal of Applied Physiology 112, no. 8 (April 15, 2012): 1393–402. http://dx.doi.org/10.1152/japplphysiol.00511.2011.
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ATP, acting through P2X2/P2X3 receptor-channel complexes, plays an important role in carotid body chemoexcitation in response to natural stimuli in the rat. Since the channels are permeable to calcium, P2X activation by ATP should induce changes in intracellular calcium ([Ca2+]i). Here, we describe a novel ex vivo approach using fluorescence [Ca2+]i imaging that allows screening of retrogradely labeled chemoafferent neurons in the petrosal ganglion of the rat. ATP-induced [Ca2+]i responses were characterized at postnatal days (P) 5–8 and P19–25. While all labeled cells showed a brisk increase in [Ca2+]i in response to depolarization by high KCl (60 mM), only a subpopulation exhibited [Ca2+]i responses to ATP. ATP (250–1,000 μM) elicited one of three temporal response patterns: fast (R1), slow (R2), and intermediate (R3). At P5–8, R2 predominated and its magnitude was attenuated 44% by the P2X1 antagonist, NF449 (10 μM), and 95% by the P2X1/P2X3/P2X2/3 antagonist, TNP-ATP (10 μM). At P19–25, R1 and R3 predominated and their magnitudes were attenuated 15% by NF449, 66% by TNP-ATP, and 100% by suramin (100 μM), a nonspecific P2 purinergic receptor antagonist. P2X1 and P2X2 protein levels in the petrosal ganglion decreased with development, while P2X3 protein levels did not change significantly. We conclude that the profile of ATP-induced P2X-mediated [Ca2+]i responses changes in the postnatal period, corresponding with changes in receptor isoform expression. We speculate that these changes may participate in the postnatal maturation of chemosensitivity.
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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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Xing, Jihong, Jian Lu, and Jianhua Li. "Augmented P2X response and immunolabeling in dorsal root ganglion neurons innervating skeletal muscle following femoral artery occlusion." Journal of Neurophysiology 109, no. 8 (April 15, 2013): 2161–68. http://dx.doi.org/10.1152/jn.01068.2012.
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The responsiveness of sensory neurons to muscle metabolites is altered under the conditions of insufficient limb blood supply in some diseases, such as peripheral artery disease. The purpose of this study was to examine ATP-induced current with activation of purinergic P2X subtypes P2X3 and P2X2/3 in dorsal root ganglion (DRG) neurons of control limbs and limbs with 24 h of femoral artery occlusion using whole cell patch-clamp methods. Also, dual-labeling immunohistochemistry was employed to determine existence of P2X3 expression in DRG neurons of thin-fiber afferents. DRG neurons from 4- to 6-wk-old rats were labeled by injecting the fluorescence tracer DiI into the hindlimb muscles 4–5 days before the recording experiments. Transient (P2X3), mixed (P2X3 and P2X2/3), and sustained (P2X2/3) current responses to α,β-methylene ATP (a P2X receptor agonist) are observed in small and medium DRG neurons, and size distribution of DRG neurons is similar in control and occluded limbs. However, the peak current amplitude of DRG neuron induced by stimulation of P2X3 and/or P2X2/3 is larger in occluded limbs than that in control limbs. Moreover, the percentage of DRG neurons with P2X3 transient currents is greater after arterial occlusion compared with control. In addition, a rapid desensitization was observed in DRG neurons with transient currents, but not with sustained currents in control and occluded groups. Furthermore, results from immunofluorescence experiments show that femoral artery occlusion primarily augments P2X3 expression within DRG neurons projecting C-fiber afferents. Overall, these findings suggest that 1) greater ATP-induced currents with activation of P2X3 and P2X2/3 are developed when hindlimb arterial blood supply is deficient under ischemic conditions and 2) increased P2X3 expression is largely observed in C-fibers of DRG neurons after hindlimb vascular insufficiency.
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Decker, Dima A., and James J. Galligan. "Cross-inhibition between nicotinic acetylcholine receptors and P2X receptors in myenteric neurons and HEK-293 cells." American Journal of Physiology-Gastrointestinal and Liver Physiology 296, no. 6 (June 2009): G1267—G1276. http://dx.doi.org/10.1152/ajpgi.00048.2009.
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The enteric nervous system (ENS) controls gut function. P2X receptors and nicotinic acetylcholine receptors (nAChRs) are ligand-gated cation channels that mediate fast synaptic excitation in the ENS. Close molecular coupling in enteric neuronal membranes contributes to a mutually inhibitory interaction between these receptors; this effect is called cross-inhibition. We studied the molecular mechanisms responsible for cross-inhibition. Whole cell patch-clamp techniques were used to measure P2X- and nAChR-mediated currents in cultured enteric neurons and HEK-293 cells. In cultured myenteric neurons, ACh (3 mM) and ATP (1 mM) coapplication evoked an inward current that was only 57 ± 6% ( P < 0.05) of the predicted current that would have occurred if the two populations of channels were activated independently. In HEK-293 cells coexpressing α3β4 nAChR/P2X2 receptors, coapplication of ATP and ACh caused a current that was 58 ± 7% of the predicted current ( P < 0.05). To test the importance of P2X subunit COOH-terminal tail length on cross-inhibition, P2X3 and P2X4 subunits, which have shorter COOH-terminal tails, were studied. Cross-inhibition with α3β4 nAChRs and P2X3 or P2X4 subunits was similar to that occurring with P2X2 subunits. P2X receptor or α3β4 nAChR desensitization did not prevent receptor cross-inhibition. These data indicate that the α3β4-P2X receptor interaction is not restricted to P2X2 subunits. In addition, active and desensitized conformations of the P2X receptor inhibit nAChR function. These molecular interactions may modulate the function of synapses that use ATP and ACh as fast synaptic transmitters in the ENS.
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Hu, Bo, Chen Yu Chiang, James W. Hu, Jonathan O. Dostrovsky, and Barry J. Sessle. "P2X Receptors in Trigeminal Subnucleus Caudalis Modulate Central Sensitization in Trigeminal Subnucleus Oralis." Journal of Neurophysiology 88, no. 4 (October 1, 2002): 1614–24. http://dx.doi.org/10.1152/jn.2002.88.4.1614.
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This study investigated the role of trigeminal subnucleus caudalis (Vc) P2X receptors in the mediation of central sensitization induced in nociceptive neurons in subnucleus oralis (Vo) by mustard oil (MO) application to the tooth pulp in anesthetized rats. MO application produced a long-lasting central sensitization reflected in neuroplastic changes (i.e., increases in neuronal mechanoreceptive field size and responses to innocuous and noxious mechanical stimuli) in Vo nociceptive neurons. Twenty minutes after MO application, the intrathecal (i.t.) administration to the rostral Vc of the selective P2X1, P2X3, and P2X2/3 receptor antagonist, 2′-(or 3′-) O-trinitrophenyl-ATP (TNP-ATP), significantly and reversibly attenuated the MO-induced central sensitization for more than 15 min; saline administration had no effect. Administration to the rostral Vc of the selective P2X1, P2X3, and P2X2/3 receptor agonist, α,β-methylene ATP (α,β-meATP, i.t.) produced abrupt and significant neuroplastic changes in Vo nociceptive neurons, followed by neuronal desensitization as evidenced by the ineffectiveness of a second i.t. application of α,β-meATP and subsequent MO application to the pulp. Administration to the rostral Vc of the selective P2X1 receptor agonist β,γ-methylene ATP (β,γ-meATP, i.t.) produced no significant neuroplastic changes per se and did not affect the subsequent MO-induced neuroplastic changes in Vo nociceptive neurons. These results suggest that P2X3 and possibly also the P2X2/3 receptor subtypes in Vc may play a role in the initiation and maintenance of central sensitization in Vo nociceptive neurons induced by MO application to the pulp.
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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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Studeny, Simon, Ali Torabi, and Margaret A. Vizzard. "P2X2 and P2X3 receptor expression in postnatal and adult rat urinary bladder and lumbosacral spinal cord." American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 289, no. 4 (October 2005): R1155—R1168. http://dx.doi.org/10.1152/ajpregu.00234.2005.
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P2X receptors mediate the effects of ATP in micturition and nociception. During postnatal maturation, a spinobulbospinal reflex and voluntary voiding replace primitive voiding reflexes. This may involve changes in neuroactive compounds and receptors in bladder reflex pathways. We examined P2X2 and P2X3 receptors in bladder and spinal cord from postnatal (P0–P36, indicating number of days) and adult Wistar rats. Western blot of whole bladders for P2X2 and P2X3 expression was performed. Immunostaining for P2X2 and P2X3 receptors in urothelium and detrusor smooth muscle whole mounts and spinal cord sections was examined. Western blot demonstrated an age-dependent decrease ( R2= 0.96, P ≤ 0.005) in P2X2 receptor expression in bladder, whereas P2X3 receptor expression in bladder peaked ( P ≤ 0.005) during P14–P21. P2X2-immunoreactivity (IR) was present in urothelial cells, suburothelial plexus, detrusor smooth muscle, and serosa at birth, with staining in urothelial cells and serosa being most predominant. With increasing postnatal age, the intensity of P2X2-IR decreased in urothelial cells but increased in suburothelial plexus. P2X3-IR increased in urothelial cells and suburothelial plexus with postnatal age, whereas staining in detrusor and serosa remained relatively constant. At birth, P2X3-IR was present in the dorsal horn, lateral collateral pathway, and dorsal commissure. With increasing age, P2X3-IR was restricted to superficial dorsal horn and lateral collateral pathway. P2X2-IR was present in ependyme cells (S-100-IR) of the central canal as early as P2. These studies demonstrate plastic expression of P2X2 and P2X3 receptors in bladder and spinal cord during early postnatal development at times coincident with appearance of mature voiding patterns.
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McCord, Jennifer L., Hirotsugu Tsuchimochi, and Marc P. Kaufman. "P2X2/3 and P2X3 receptors contribute to the metaboreceptor component of the exercise pressor reflex." Journal of Applied Physiology 109, no. 5 (November 2010): 1416–23. http://dx.doi.org/10.1152/japplphysiol.00774.2010.
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The exercise pressor reflex is due to activation of thin fiber afferents within contracting muscle. These afferents are in part stimulated by ATP activation of purinergic 2X (P2X) receptors during contraction. Which of the P2X receptors contribute to the reflex is unknown; however, P2X2/3 and P2X3 receptor subtypes are good candidates because they are located on thin fiber afferents and are involved in sensory neurotransmission. To determine if P2X2/3 and P2X3 receptors evoke the metabolic component of the exercise pressor reflex, we examined the effect of two P2X2/3 and P2X3 antagonists, A-317491 (10 mg/kg) and RO-3 (10 mg/kg), on the pressor response to injections of α,β-methylene ATP (α,β-MeATP; 50 μg/kg), freely perfused static contraction, contraction of the triceps surae muscles while the circulation was occluded, and postcontraction circulatory occlusion in decerebrate cats. We found that the antagonists reduced the pressor response to α,β-MeATP injection (before Δ 20 ± 3 mmHg; drug Δ 11 ± 3 mmHg; P < 0.05), suggesting the antagonists were effective in blocking P2X2/3 and P2X3 receptors. P2X2/3 and P2X3 receptor blockade reduced the pressor response to freely perfused contraction (before Δ 33 ± 5 mmHg; drug Δ 15 ± 5 mmHg; P < 0.05), contraction with the circulation occluded (before Δ 52 ± 7 mmHg; drug Δ 20 ± 4 mmHg; P < 0.05), and during postcontraction circulatory occlusion (before Δ 15 ± 1 mmHg; drug Δ 5 ± 1 mmHg; P < 0.05). Our findings suggest that P2X2/3 and P2X3 receptors contribute to the metabolic component of the exercise pressor reflex in decerebrate cats.
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Zhuang, Jianguo, Xiuping Gao, Wan Wei, Amir Pelleg, and Fadi Xu. "Intralaryngeal application of ATP evokes apneic response mainly via acting on P2X3 (P2X2/3) receptors of the superior laryngeal nerve in postnatal rats." Journal of Applied Physiology 131, no. 3 (September 1, 2021): 986–96. http://dx.doi.org/10.1152/japplphysiol.00091.2021.
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Aerosolized ATP induces cough and bronchoconstriction via activating P2X3 and P2X2/3 receptors (P2X3R and P2X2/3R) localized on vagal pulmonary sensory fibers. The superior laryngeal nerve (SLN), particularly SLN C-fibers (SLCFs), is involved in generating apnea, hypertension, and bradycardia. This study demonstrates for the first time that either ATP or α,β-mATP applied onto the laryngeal mucosa elicit these cardiorespiratory responses predominately through the activation of P2X3R-P2X2/3R localized on SLCFs.
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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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Burgard, Edward C., Wende Niforatos, Tim van Biesen, Kevin J. Lynch, Edward Touma, Randy E. Metzger, Elizabeth A. Kowaluk, and Michael F. Jarvis. "P2X Receptor–Mediated Ionic Currents in Dorsal Root Ganglion Neurons." Journal of Neurophysiology 82, no. 3 (September 1, 1999): 1590–98. http://dx.doi.org/10.1152/jn.1999.82.3.1590.
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Nociceptive neurons in the dorsal root ganglia (DRG) are activated by extracellular ATP, implicating P2X receptors as potential mediators of painful stimuli. However, the P2X receptor subtype(s) underlying this activity remain in question. Using electrophysiological techniques, the effects of P2X receptor agonists and antagonists were examined on acutely dissociated adult rat lumbar DRG neurons. Putative P2X-expressing nociceptors were identified by labeling neurons with the lectin IB4. These neurons could be grouped into three categories based on response kinetics to extracellularly applied ATP. Some DRG responses (slow DRG) were relatively slowly activating, nondesensitizing, and activated by the ATP analogue α,β-meATP. These responses resembled those recorded from 1321N1 cells expressing recombinant heteromultimeric rat P2X2/3 receptors. Other responses (fast DRG) were rapidly activating and desensitized almost completely during agonist application. These responses had properties similar to those recorded from 1321N1 cells expressing recombinant rat P2X3 receptors. A third group (mixed DRG) activated and desensitized rapidly (P2X3-like), but also had a slow, nondesensitizing component that functionally prolonged the current. Like the fast component, the slow component was activated by both ATP and α,β-meATP and was blocked by the P2X antagonist TNP-ATP. But unlike the fast component, the slow component could follow high-frequency activation by agonist, and its amplitude was potentiated under acidic conditions. These characteristics most closely resemble those of rat P2X2/3 receptors. These data suggest that there are at least two populations of P2X receptors present on adult DRG nociceptive neurons, P2X3 and P2X2/3. These receptors are expressed either separately or together on individual neurons and may play a role in the processing of nociceptive information from the periphery to the spinal cord.
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Belleannée, Clémence, Nicolas Da Silva, Winnie W. C. Shum, Dennis Brown, and Sylvie Breton. "Role of purinergic signaling pathways in V-ATPase recruitment to apical membrane of acidifying epididymal clear cells." American Journal of Physiology-Cell Physiology 298, no. 4 (April 2010): C817—C830. http://dx.doi.org/10.1152/ajpcell.00460.2009.
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Extracellular purinergic agonists regulate a broad range of physiological functions via P1 and P2 receptors. Using the epididymis as a model system in which luminal acidification is essential for sperm maturation and storage, we show here that extracellular ATP and its hydrolysis product adenosine trigger the apical accumulation of vacuolar H+-ATPase (V-ATPase) in acidifying clear cells. We demonstrate that the epididymis can hydrolyze luminal ATP into other purinergic agonists such as ADP via the activity of nucleotidases located in the epididymal fluid and in the apical membrane of epithelial cells. Alkaline phosphatase activity and abundant ecto-5′-nucleotidase protein were detected in the apical pole of principal cells. In addition, we show that nine nucleotidase genes ( Nt5e, Alpl, Alpp, Enpp1, 2, and 3, and Entpd 2, 4, and 5), seven ATP P2 receptor genes (P2X1, P2X2, P2X3, P2X4, P2X6, P2Y2, P2Y5), and three adenosine P1 receptor genes (A1, A2B, and A3) are expressed in epithelial cells isolated by laser cut microdissection (LCM). The calcium chelator BAPTA-AM abolished the apical V-ATPase accumulation induced by ATP, supporting the contribution of P2X or P2Y in this response. The PKA inhibitor myristoylated protein kinase inhibitor (mPKI) inhibited adenosine-dependent V-ATPase apical accumulation, indicating the participation of the P1 A2B receptor. Altogether, these results suggest that the activation of P1 and P2 purinergic receptors by ATP and adenosine might play a significant role in luminal acidification in the epididymis, a process that is crucial for the establishment of male fertility.
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Doctor, R. Brian, Thomas Matzakos, Ryan McWilliams, Sylene Johnson, Andrew P. Feranchak, and J. Gregory Fitz. "Purinergic regulation of cholangiocyte secretion: identification of a novel role for P2X receptors." American Journal of Physiology-Gastrointestinal and Liver Physiology 288, no. 4 (April 2005): G779—G786. http://dx.doi.org/10.1152/ajpgi.00325.2004.
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The P2X family of ligand-gated cation channels is comprised of seven distinct isoforms activated by binding of extracellular purines. Although originally identified in neurons, there is increasing evidence for expression of P2X receptors in epithelia as well. Because ATP is released by both hepatocytes and cholangiocytes, these studies were performed to evaluate whether P2X receptors are present in cholangiocytes and contribute to local regulation of biliary secretion and bile formation. RT-PCR of cDNA from cultured normal rat cholangiocytes detected transcripts for P2X receptors 2, 3, 4, and 6; products from P2X3 and P2X4 were robust and always detectable. In cholangiocyte lysates, P2X4 protein was readily detected, and immunohistochemical staining of intact rat liver revealed P2X4 protein concentrated in intrahepatic bile ducts. To assess the functional significance of P2X4, isolated Mz-ChA-1 cells were exposed to the P2X4-preferring agonist 2′,3′-O-(4-benzoyl-benzoyl)-ATP (BzATP), which activated inward currents of −18.2 + 3.0 pA/pF. In cholangiocyte monolayers, BzATP but not P2X3 agonists elicited robust Cl−secretory responses (short-circuit current) when applied to either the apical (Δ Isc22.1 ± 3.3 μA) or basolateral (18.5 ± 1.6 μA) chamber, with half-maximal stimulation at ∼10 μM and ∼1 μM, respectively. The response to BzATP was unaffected by suramin (not significant) and was inhibited by Cu2+( P < 0.01). These studies provide molecular and biochemical evidence for the presence of P2X receptors in cholangiocytes. Functional studies indicate that P2X4 is likely the primary isoform involved, representing a novel and functionally important component of the purinergic signaling complex modulating biliary secretion.
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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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Kim, Chang-Hoon, Sung-Shik Kim, Jae Young Choi, Ji-Hyun Shin, Jin Young Kim, Wan Namkung, Jeung-Gweon Lee, Min Goo Lee, and Joo-Heon Yoon. "Membrane-specific expression of functional purinergic receptors in normal human nasal epithelial cells." American Journal of Physiology-Lung Cellular and Molecular Physiology 287, no. 4 (October 2004): L835—L842. http://dx.doi.org/10.1152/ajplung.00285.2003.
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Extracellular purines and pyrimidines regulate various physiological responses via the cell surface receptors known as purinoreceptors and may exert autocrine or paracrine effects on ion transport, fluid transport, ciliary beat frequency, and mucin secretion. Therefore, this study aims to investigate the expression patterns of the purinoreceptors in normal human nasal epithelial (NHNE) cells. In RT-PCR, the mRNAs for several P2X (P2X3, P2X4, P2X7) and P2Y (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12) receptors were identified in NHNE cells. Functional localizations of P2 receptors were investigated by measuring intracellular calcium concentration ([Ca2+]i) increases in membrane-specific manner using a double-perfusion chamber. Absence of the responses of αβ-methylene ATP and 2-methylthio-ATP excluded functionally active P2X3, P2X4, and P2Y1 receptors as far as [Ca2+]i increase is concerned. Applications with ATP and UTP revealed that luminal membranes of NHNE cells express P2Y2 and P2Y6 receptors and basolateral membranes express P2Y2 receptor. Expressions of P2Y2 and P2Y6 receptors in NHNE cells were further verified by immunoblotting using specific antibodies. In addition, the results with 2,3-O-(4-benzoyl)-benzoyl-ATP indicate that the P2Y11 receptor may be present on the luminal side. In conclusion, the NHNE cells express functionally active P2Y2, P2Y6, and P2Y11 receptors in a membrane-specific pattern, which may play an important role in the control of mucin and fluid secretion in NHNE cells.
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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.
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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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Shabir, Saqib, William Cross, Lisa A. Kirkwood, Joanna F. Pearson, Peter A. Appleby, Dawn Walker, Ian Eardley, and Jennifer Southgate. "Functional expression of purinergic P2 receptors and transient receptor potential channels by the human urothelium." American Journal of Physiology-Renal Physiology 305, no. 3 (August 1, 2013): F396—F406. http://dx.doi.org/10.1152/ajprenal.00127.2013.
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In addition to its role as a physical barrier, the urothelium is considered to play an active role in mechanosensation. A key mechanism is the release of transient mediators that activate purinergic P2 receptors and transient receptor potential (TRP) channels to effect changes in intracellular Ca2+. Despite the implied importance of these receptors and channels in urothelial tissue homeostasis and dysfunctional bladder disease, little is known about their functional expression by the human urothelium. To evaluate the expression and function of P2X and P2Y receptors and TRP channels, the human ureter and bladder were used to separate urothelial and stromal tissues for RNA isolation and cell culture. RT-PCR using stringently designed primer sets was used to establish which P2 and TRP species were expressed at the transcript level, and selective agonists/antagonists were used to confirm functional expression by monitoring changes in intracellular Ca2+ and in a scratch repair assay. The results confirmed the functional expression of P2Y4 receptors and excluded nonexpressed receptors/channels (P2X1, P2X3, P2X6, P2Y6, P2Y11, TRPV5, and TRPM8), while a dearth of specific agonists confounded the functional validation of expressed P2X2, P2X4, P2Y1, P2Y2, TRPV2, TRPV3, TRPV6 and TRPM7 receptors/channels. Although a conventional response was elicited in control stromal-derived cells, the urothelial cell response to well-characterized TRPV1 and TRPV4 agonists/antagonists revealed unexpected anomalies. In addition, agonists that invoked an increase in intracellular Ca2+ promoted urothelial scratch repair, presumably through the release of ATP. The study raises important questions about the ligand selectivity of receptor/channel targets expressed by the urothelium. These pathways are important in urothelial tissue homeostasis, and this opens the possibility of selective drug targeting.
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Wang, Lizhao, Dan Feng, Huanhuan Yan, Zhongping Wang, and Lei Pei. "Comparative Analysis of P2X1, P2X2, P2X3, and P2X4 Receptor Subunits in Rat Nodose Ganglion Neurons." PLoS ONE 9, no. 5 (May 5, 2014): e96699. http://dx.doi.org/10.1371/journal.pone.0096699.
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Sterle, Igor, Daša Zupančič, and Rok Romih. "Correlation between Urothelial Differentiation and Sensory Proteins P2X3, P2X5, TRPV1, and TRPV4 in Normal Urothelium and Papillary Carcinoma of Human Bladder." BioMed Research International 2014 (2014): 1–9. http://dx.doi.org/10.1155/2014/805236.
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Terminal differentiation of urothelium is a prerequisite for blood-urine barrier formation and enables normal sensory function of the urinary bladder. In this study, urothelial differentiation of normal human urothelium and of low and high grade papillary urothelial carcinomas was correlated with the expression and localization of purinergic receptors (P2X3, and P2X5) and transient receptor potential vanilloid channels (TRPV1, and TRPV4). Western blotting and immunofluorescence of uroplakins together with scanning electron microscopy of urothelial apical surface demonstrated terminal differentiation of normal urothelium, partial differentiation of low grade carcinoma, and poor differentiation of high grade carcinoma. P2X3 was expressed in normal urothelium as well as in low grade carcinoma and in both cases immunolabeling was stronger in the superficial cells. P2X3 expression decreased in high grade carcinoma. P2X5 expression was detected in normal urothelium and in high grade carcinoma, while in low grade carcinoma its expression was diminished. The expression of TRPV1 decreased in low grade and even more in high grade carcinoma when compared with normal urothelium, while TRPV4 expression was unchanged in all samples. Our results suggest that sensory proteins P2X3 and TRPV1 are in correlation with urothelial differentiation, while P2X5 and TRPV4 have unique expression patterns.
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Yu, Jie, Junying Du, Junfan Fang, Yingjun Liu, Xuaner Xiang, Yi Liang, Xiaomei Shao, and Jianqiao Fang. "The interaction between P2X3 and TRPV1 in the dorsal root ganglia of adult rats with different pathological pains." Molecular Pain 17 (January 2021): 174480692110113. http://dx.doi.org/10.1177/17448069211011315.
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Peripheral inflammatory and neuropathic pain are closely related to the activation of purinergic receptor P2X ligand-gated ion channel 3 (P2X3) and transient receptor potential vanilloid 1 (TRPV1), but the interaction between P2X3 and TRPV1 in different types of pathological pain has rarely been reported. In this study, complete Freund’s adjuvant (CFA)-induced inflammatory pain and spared nerve injury (SNI)-induced neuropathic pain models were established in adult rats. The interactions between P2X3 and TRPV1 in the dorsal root ganglion were observed by pharmacological, co-immunoprecipitation, immunofluorescence and whole-cell patch-clamp recording assays. TRPV1 was shown to promote the induction of spontaneous pain caused by P2X3 in the SNI model, but the induction of spontaneous pain behaviour by TRPV1 was not completely dependent on P2X3 in vivo. In both the CFA and SNI models, the activation of peripheral P2X3 enhanced the effect of TRPV1 on spontaneous pain, while the inhibition of peripheral TRPV1 reduced the induction of spontaneous pain by P2X3 in the CFA model. TRPV1 and P2X3 had inhibitory effects on each other in the inflammatory pain model. During neuropathic pain, P2X3 facilitated the function of TRPV1, while TRPV1 had an inhibitory effect on P2X3. These results suggest that the mutual effects of P2X3 and TRPV1 differ in cases of inflammatory and neuropathic pain in rats.
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Tsuzuki, Kenzo, Ariel Ase, Philippe Séguéla, Terumasa Nakatsuka, Cong-Yi Wang, Jin-Xiong She, and Jianguo G. Gu. "TNP-ATP-Resistant P2X Ionic Current on the Central Terminals and Somata of Rat Primary Sensory Neurons." Journal of Neurophysiology 89, no. 6 (June 2003): 3235–42. http://dx.doi.org/10.1152/jn.01171.2002.
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P2X receptors have been suggested to be expressed on the central terminals of Aδ-afferent fibers innervating dorsal horn lamina V and play a role in modulating sensory synaptic transmission. These P2X receptors have been widely thought to be P2X2+3 receptors. However, we have recently found that P2X receptor-mediated modulation of sensory transmission in lamina V is not inhibited by trinitrophenyl-adenosine triphosphate (TNP-ATP), a potent antagonist of P2X1, P2X3 homomers, and P2X2+3 heteromers. To provide direct evidence for the presence of TNP-ATP-resistant P2X receptors on primary afferent fibers, we examined α,β-methylene-ATP (αβmeATP)-evoked currents and their sensitivity to TNP-ATP in rat dorsal root ganglion (DRG) neurons. αβmeATP evoked fast currents, slow currents, and mixed currents that contained both fast and slow current-components. Fast currents and fast current components in the mixed currents were both completely inhibited by 0.1 μM TNP-ATP ( n = 14). Both slow currents and slow-current components in the mixed currents showed broad spectrum of sensitivity to 1 μM TNP-ATP, ranging from complete block (TNP-ATP-sensitive) to little block (TNP-ATP-resistant). TNP-ATP-resistant currents evoked by 10 μM αβmeATP could be largely inhibited by 10 μM iso-pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid. Cells with P2X currents that were highly resistant to TNP-ATP were found to be insensitive to capsaicin. These results suggest that TNP-ATP-resistant P2X receptor subtypes are expressed on capsaicin-insensitive Aδ-afferent fibers and play a role in modulating sensory transmission to lamina V neurons.
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Niane, Lalah M., David F. Donnelly, Vincent Joseph, and Aida Bairam. "Ventilatory and carotid body chemoreceptor responses to purinergic P2X receptor antagonists in newborn rats." Journal of Applied Physiology 110, no. 1 (January 2011): 83–94. http://dx.doi.org/10.1152/japplphysiol.00871.2010.
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Adenosine triphosphate, acting through purinergic P2X receptors, has been shown to stimulate ventilation and increase carotid body chemoreceptor activity in adult rats. However, its role during postnatal development of the ventilatory response to hypoxia is yet unknown. Using whole body plethysmography, we measured ventilation in normoxia and in moderate hypoxia (12% fraction of inspired O2, 20 min) before and after intraperitoneal injection of suramin (P2X2 and P2X3 receptor antagonist, 40 mg/kg) in 4-, 7-, 12-, and 21-day-old rats. Suramin reduced baseline breathing (∼20%) and the response to hypoxia (∼30%) in all rats, with a relatively constant effect across ages. We then tested the effect of the specific P2X3 antagonist, A-317491 (150 mg/kg), in rats aged 4, 7, and 21 days. As with suramin, A-317491 reduced baseline ventilation (∼55%) and the hypoxic response (∼40%) at all ages studied. Single-unit carotid body chemoreceptor activity was recorded in vitro in 4-, 7-, and 21-day-old rats. Suramin (100 μM) and A-317491 (10 μM) significantly depressed the sinus nerve chemosensory discharge rate (∼80%) in normoxia (Po2 ∼150 Torr) and hypoxia (Po2 ∼60 Torr), and this decrease was constant across ages. We conclude that, in newborn rats, P2X purinergic receptors are involved in the regulation of breathing under basal and hypoxic condition, and P2X3-containing receptors play a major role in carotid body function. However, these effects are not age dependent within the age range studied.
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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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Wang, Jin, Yao Wang, Wen-Wen Cui, Yichen Huang, Yang Yang, Yan Liu, Wen-Shan Zhao, et al. "Druggable negative allosteric site of P2X3 receptors." Proceedings of the National Academy of Sciences 115, no. 19 (April 19, 2018): 4939–44. http://dx.doi.org/10.1073/pnas.1800907115.
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Allosteric modulation provides exciting opportunities for drug discovery of enzymes, ion channels, and G protein-coupled receptors. As cation channels gated by extracellular ATP, P2X receptors have attracted wide attention as new drug targets. Although small molecules targeting P2X receptors have entered into clinical trials for rheumatoid arthritis, cough, and pain, negative allosteric modulation of these receptors remains largely unexplored. Here, combining X-ray crystallography, computational modeling, and functional studies of channel mutants, we identified a negative allosteric site on P2X3 receptors, fostered by the left flipper (LF), lower body (LB), and dorsal fin (DF) domains. Using two structurally analogous subtype-specific allosteric inhibitors of P2X3, AF-353 and AF-219, the latter being a drug candidate under phase II clinical trials for refractory chronic cough and idiopathic pulmonary fibrosis, we defined the molecular interactions between the drugs and receptors and the mechanism by which allosteric changes in the LF, DF, and LB domains modulate ATP activation of P2X3. Our detailed characterization of this druggable allosteric site should inspire new strategies to develop P2X3-specific allosteric modulators for clinical use.
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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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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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Bölcskei, Hedvig, and Bence Farkas. "P2X3 and P2X2/3 receptor antagonists." Pharmaceutical Patent Analyst 3, no. 1 (January 2014): 53–64. http://dx.doi.org/10.4155/ppa.13.70.
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Shigematsu, Yasuhide, Yukio Shimoda, and Makoto Kaneda. "Distribution of immunoreactivity for P2X3, P2X5, and P2X6-purinoceptors in mouse retina." Journal of Molecular Histology 38, no. 4 (June 29, 2007): 369–71. http://dx.doi.org/10.1007/s10735-007-9107-4.
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Liu, Jiahao, Jialiu David Li, Jian Lu, Jihong Xing, and Jianhua Li. "Contribution of nerve growth factor to upregulation of P2X3 expression in DRG neurons of rats with femoral artery occlusion." American Journal of Physiology-Heart and Circulatory Physiology 301, no. 3 (September 2011): H1070—H1079. http://dx.doi.org/10.1152/ajpheart.00188.2011.
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Femoral artery occlusion augments the sympathetic nerve and pressor responses to muscle contraction and muscle metabolites injected into the arterial blood supply of the hindlimb muscles in rats. The underlying mechanism by which these reflex responses are enhanced after muscle vascular insufficiency is unclear. Purinergic P2X3 receptor has been reported to contribute to the metabolic component of the exercise pressor reflex. Thus the purpose of this study was to examine if chronic femoral occlusion would alter the expression of P2X3 in dorsal root ganglion (DRG) neurons of rats. Also, P2X3-mediated sympathetic responsiveness was examined after femoral occlusion. In addition, the role played by nerve growth factor (NGF) in regulating the expression and response of P2X3 was examined. Western blot analysis showed that 24 h of femoral ligation increased the levels of P2X3 (optical density: 0.93 ± 0.07 in control and 1.37 ± 0.10 after occlusion; P < 0.05 vs. control). The fluorescence immunohistochemistry further demonstrated that the occlusion elevated P2X3 expression in DRG neurons (percentage of P2X3-positive cells: 33 ± 3% in control and 51 ± 3% in occlusion; P < 0.05 vs. control). Furthermore, the results showed that responses of renal sympathetic nerve activity and blood pressure to stimulation of P2X were greater in occluded rats than responses in control rats by injection of α,β-methylene ATP into the arterial blood supply of the hindlimb muscle. Finally, infusion of NGF in the hindlimb muscles of healthy rats increased P2X3 (optical density: 0.98 ± 0.12 in control and 1.37 ± 0.16 with NGF; P < 0.05 vs. control). The pressor response to injection of α,β-methylene ATP was increased in the rats with NGF infusion. Likewise, blocking NGF attenuated exaggeration of the reflex response induced by α,β-methylene ATP in occluded rats. The findings of this study suggest that the levels of P2X3 in primary afferent neurons are upregulated as the blood supply to the hindlimb is deficient under ischemic conditions, leading to augmentation of the muscle reflex. NGF is closely related to increases in P2X3 receptor expression and response.
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Liang, Lihua, Akos Zsembery, and Erik M. Schwiebert. "RNA interference targeted to multiple P2X receptor subtypes attenuates zinc-induced calcium entry." American Journal of Physiology-Cell Physiology 289, no. 2 (August 2005): C388—C396. http://dx.doi.org/10.1152/ajpcell.00491.2004.
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A postulated therapeutic avenue in cystic fibrosis (CF) is activation of Ca2+-dependent Cl− channels via stimulation of Ca2+ entry from extracellular solutions independent of CFTR functional status. We have shown that extracellular zinc and ATP induce a sustained increase in cytosolic Ca2+ in human airway epithelial cells that translates into stimulation of sustained secretory Cl− transport in non-CF and CF human and mouse airway epithelial cells, cell monolayers, and nasal mucosa. On the basis of these studies, the Ca2+ entry channels most likely involved were P2X purinergic receptor channels. In the present study, molecular and biochemical data show coexpression of P2X4, P2X5, and P2X6 subtypes in non-CF (16HBE14o−) and CF (IB3-1) human bronchial epithelial cells. Other P2X receptor Ca2+ entry channel subtypes are expressed rarely or not at all in airway epithelia, epithelial cell models from other CF-relevant tissues, or vascular endothelia. Novel transient lipid transfection-mediated delivery of small interference RNA fragments specific to P2X4 and P2X6 (but not P2X5) into IB3-1 CF human airway epithelial cells inhibited extracellular zinc- and ATP-induced Ca2+ entry markedly in fura-2 Ca2+ measurements and “knocked down” protein by >65%. These data suggest that multiple P2X receptor Ca2+ entry channel subtypes are expressed in airway epithelia. P2X4 and P2X6 may coassemble on the airway surface as targets for possible therapeutics for CF independent of CFTR genotype.
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Ding, Shaojie, Qin Yu, Jianzhang Wang, Libo Zhu, Tiantian Li, Xinyue Guo, and Xinmei Zhang. "Activation of ATF3/AP-1 signaling pathway is required for P2X3-induced endometriosis pain." Human Reproduction 35, no. 5 (April 18, 2020): 1130–44. http://dx.doi.org/10.1093/humrep/deaa061.
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Abstract STUDY QUESTION Does P2X ligand-gated ion channel 3 (P2X3) play a role in endometriosis pain? SUMMARY ANSWER Upregulation of P2X3 in dorsal root ganglia (DRG) tissues via the activating transcription factor 3 (ATF3)/activator protein (AP)-1 pathway contributed to endometriosis-associated hyperalgesia, which could be attenuated by the chitosan oligosaccharide stearic acid (CSOSA)/liposomes (LPs)/SP600125 delivery system. WHAT IS KNOWN ALREADY Infiltrating nerve fibers and elevated nociceptors in endometriotic lesions are associated with endometriosis pain. P2X3 has been demonstrated to play an important role in neuropathic pain. STUDY DESIGN, SIZE, DURATION A rat model of endometriosis was used to investigate the signaling pathways involved in P2X3-induced pain. PARTICIPANTS/MATERIALS, SETTING, METHODS Degrees of hyperalgesia, endogenous adenosine 5′-triphosphate (ATP) contents and P2X3 expression levels in endometriotic lesions and DRG tissues were detected in a rat model of endometriosis. The expression levels of ATF3 and P2X3 were measured using qRT-PCR, western blot analysis and immunofluorescence analysis after adenosine 5′-diphosphate (ADP) exposure in DRG cells. Plasmids encoding ATF3 and its siRNA were used to investigate the role of ATF3 on ADP-induced P2X3 upregulation. The activity of ATF binding to the P2X3 promoter was evaluated by using chromatin immunoprecipitation (CHIP) and luciferase assays. SP600125, an inhibitor of c-JUN N-terminal kinase, was wrapped in CSOSA/LPs delivery system and its inhibitory effects on ADP-induced upregulation of P2X3 in DRG cells and endometriosis-induced hyperalgesia in rats were tested. MAIN RESULTS AND THE ROLE OF CHANCE The concentrations of endogenous ATP and expression levels of P2X3 were significantly increased in both endometriotic lesions and DRG tissues in endometriosis rat models and were found to be positively correlated with the severity of hyperalgesia. In DRG cells, P2X3 expression levels were elevated by ADP stimulation, but dramatically inhibited by blocking ATF3 with its siRNA and SP600125. CHIP and luciferase assay showed that ADP increased the binding of ATF3 to the P2X3 promoter, resulting in an increase in P2X3 expression levels. In the CSOSA/LPs/SP600125 delivery system, the drug could be effectively concentrated in endometriotic lesions, and it could alleviate endometriosis-induced hyperalgesia, reduce the size of endometriotic lesions and attenuate upregulated P2X3 expression levels in endometriosis rat models. LARGE SCALE DATA N/A LIMITATIONS, REASONS FOR CAUTION Changes in the sensitivity and function of P2X3 caused by endometriosis need to be further investigated. WIDER IMPLICATIONS OF THE FINDINGS This study indicates that ATP and the P2X3 receptor are involved in endometriosis pain, thus providing a novel therapeutic approach for the treatment of endometriosis pain by targeting the P2X3 receptor. STUDY FUNDING/COMPETING INTEREST(S) This work was funded by National Key R&D Program of China (Grant No. 2017YFC1001202) and National Natural Science Foundation of China (Grant Nos. 81974225, 81671429 and 81471433). There are no competing interests.
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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.
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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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Xing, Jihong, and Jianhua Li. "Bradykinin Contributes to Sympathetic and Pressor Responses Evoked by Activation of Skeletal Muscle Afferents P2X in Heart Failure." Cellular Physiology and Biochemistry 39, no. 6 (2016): 2101–9. http://dx.doi.org/10.1159/000447906.
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Background/Aims: Published data suggest that purinergic P2X receptors of muscle afferent nerves contribute to the enhanced sympathetic nervous activity (SNA) and blood pressure (BP) responses during static exercise in heart failure (HF). In this study, we examined engagement of bradykinin (BK) in regulating responses of SNA and BP evoked by P2X stimulation in rats with HF. We further examined cellular mechanisms responsible for BK. We hypothesized that BK potentiates P2X currents of muscle dorsal root ganglion (DRG) neurons, and this effect is greater in HF due to upregulation of BK kinin B2 and P2X3 receptor. As a result, BK amplifies muscle afferents P2X-mediated SNA and BP responses. Methods: Renal SNA and BP responses were recorded in control rats and rats with HF. Western Blot analysis and patch-clamp methods were employed to examine the receptor expression and function of DRG neurons involved in the effects of BK. Results: BK injected into the arterial blood supply of the hindlimb muscles heightened the reflex SNA and BP responses induced by P2X activation with α,β-methylene ATP to a greater degree in HF rats. In addition, HF upregulated the protein expression of kinin B2 and P2X3 in DRG and the prior application of BK increased the magnitude of α,β-methylene ATP-induced currents in muscle DRG neurons from HF rats. Conclusion: BK plays a facilitating role in modulating muscle afferent P2X-engaged reflex sympathetic and pressor responses. In HF, P2X responsivness is augmented due to increases in expression of kinin B2 and P2X3 receptors and P2X current activity.
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Kwong, Kevin, Marian Kollarik, Christina Nassenstein, Fei Ru, and Bradley J. Undem. "P2X2 receptors differentiate placodal vs. neural crest C-fiber phenotypes innervating guinea pig lungs and esophagus." American Journal of Physiology-Lung Cellular and Molecular Physiology 295, no. 5 (November 2008): L858—L865. http://dx.doi.org/10.1152/ajplung.90360.2008.
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The lungs and esophagus are innervated by sensory neurons with somata in the nodose, jugular, and dorsal root ganglion. These sensory ganglia are derived from embryonic placode (nodose) and neural crest tissues (jugular and dorsal root ganglia; DRG). We addressed the hypothesis that the neuron's embryonic origin (e.g., placode vs. neural crest) plays a greater role in determining particular aspects of its phenotype than the environment in which it innervates (e.g., lungs vs. esophagus). This hypothesis was tested using a combination of extracellular and patch-clamp electrophysiology and single-cell RT-PCR from guinea pig neurons. Nodose, but not jugular C-fibers innervating the lungs and esophagus, responded to α,β-methylene ATP with action potential discharge that was sensitive to the P2X3 (P2X2/3) selective receptor antagonist A-317491. The somata of lung- and esophagus-specific sensory fibers were identified using retrograde tracing with a fluorescent dye. Esophageal- and lung-traced neurons from placodal tissue (nodose neurons) responded similarly to α,β-methylene ATP (30 μM) with a large sustained inward current, whereas in neurons derived from neural crest tissue (jugular and DRG neurons), the same dose of α,β-methylene ATP resulted in only a transient rapidly inactivating current or no detectable current. It has been shown previously that only activation of P2X2/3 heteromeric receptors produce sustained currents, whereas homomeric P2X3 receptor activation produces a rapidly inactivating current. Consistent with this, single-cell RT-PCR analysis revealed that the nodose ganglion neurons innervating the lungs and esophagus expressed mRNA for P2X2 and P2X3 subunits, whereas the vast majority of jugular and dorsal root ganglia innervating these tissues expressed only P2X3 mRNA with little to no P2X2 mRNA expression. We conclude that the responsiveness of C-fibers innervating the lungs and esophagus to ATP and other purinergic agonists is determined more by their embryonic origin than by the environment of the tissue they ultimately innervate.
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Kitahara, Seiko, Megumi Yamashita, and Yoshimi Ikemoto. "Effects of pentobarbital on purinergic P2X receptors of rat dorsal root ganglion neurons." Canadian Journal of Physiology and Pharmacology 81, no. 12 (December 1, 2003): 1085–91. http://dx.doi.org/10.1139/y03-111.
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Purinergic P2X receptors are ligand-gated ion channels that are activated by extracellular adenosine triphosphate (ATP) and are widely expressed not only in the central and peripheral nervous system but also in tissues throughout the body, playing an important role in the transfer of nociceptive information. Since the influence of barbiturates on P2X receptor subtypes is not known, we studied the effects of pentobarbital sodium (PB) on ATP responses in dorsal root ganglion (DRG) neurons. DRG neurons were dissected from 10- to 14-day-old rats and dissociated after enzyme treatment. Electrical measurements were performed using the nystatin-perforated patch recording mode under voltage-clamp conditions. Drugs were applied using the Y-tube method. ATP evoked three types of inward current at –60 mV: fast desensitizing, slow desensitizing, and mixed. The fast-type current was attributed to activation of P2X3 subtype and the slow type to the P2X2 subtype. PB suppressed the fast-type current in a concentration-dependent manner, while the slow type was slightly reduced. A noncompetitive inhibition was suggested by a downward shift of the ATP concentration–response curves. The current–voltage relationships showed inward rectification, and the extent of suppression was not affected by the holding potential. The reduction was greater in external solutions of higher pH. PB had subtype-specific effects on P2X receptors. The ionized form is likely to be responsible for the suppression of the P2X3 receptor current, which may result in a reduction of the excitability of central and peripheral neurons and may contribute to the anesthetic and analgesic actions of the agent.Key words: pentobarbital sodium, ATP, P2X receptors, rat DRG neurons, patch clamp.
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Trapero, Carla, and Mireia Martín-Satué. "Purinergic Signaling in Endometriosis-Associated Pain." International Journal of Molecular Sciences 21, no. 22 (November 12, 2020): 8512. http://dx.doi.org/10.3390/ijms21228512.
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Endometriosis is an estrogen-dependent gynecological disease, with an associated chronic inflammatory component, characterized by the presence of endometrial tissue outside the uterine cavity. Its predominant symptom is pain, a condition notably altering the quality of life of women with the disease. This review is intended to exhaustively gather current knowledge on purinergic signaling in endometriosis-associated pain. Altered extracellular ATP hydrolysis, due to changes in ectonucleotidase activity, has been reported in endometriosis; the resulting accumulation of ATP in the endometriotic microenvironment points to sustained activation of nucleotide receptors (P2 receptors) capable of generating a persistent pain message. P2X3 receptor, expressed in sensory neurons, mediates nociceptive, neuropathic, and inflammatory pain, and is enrolled in endometriosis-related pain. Pharmacological inhibition of P2X3 receptor is under evaluation as a pain relief treatment for women with endometriosis. The role of other ATP receptors is also discussed here, e.g., P2X4 and P2X7 receptors, which are involved in inflammatory cell–nerve and microglia–nerve crosstalk, and therefore in inflammatory and neuropathic pain. Adenosine receptors (P1 receptors), by contrast, mainly play antinociceptive and anti-inflammatory roles. Purinome-targeted drugs, including nucleotide receptors and metabolizing enzymes, are potential non-hormonal therapeutic tools for the pharmacological management of endometriosis-related pain.
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Jabs, Ronald, Elke Guenther, Katja Marquordt, and Thomas H. Wheeler-Schilling. "Evidence for P2X3, P2X4, P2X5 but not for P2X7 containing purinergic receptors in Müller cells of the rat retina." Molecular Brain Research 76, no. 2 (March 2000): 205–10. http://dx.doi.org/10.1016/s0169-328x(99)00339-3.
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