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Zhang Y, Zhang F, Liu Z, Li M, Wu G, Li H. P2RX1-blocked neutrophils induce CD8 + T cell dysfunction and affect the immune escape of gastric cancer cells. Cell Immunol 2025; 408:104901. [PMID: 39675308 DOI: 10.1016/j.cellimm.2024.104901] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/19/2024] [Revised: 11/28/2024] [Accepted: 11/30/2024] [Indexed: 12/17/2024]
Abstract
BACKGROUND Gastric cancer (GC) is one of the deadly malignancies of the gastrointestinal tract. Research has confirmed the linkage of P2RX1 with immune cell activation and tumor progression. This project focused on the impact of P2RX1 level in neutrophils on the efficacy of immune checkpoint inhibitor (ICI) treatment in GC. METHODS Blood samples from 23 GC patients eligible for camrelizumab treatment were collected. Flow cytometry was carried out to analyze the proportion of P2RX1 in neutrophils. IHC was utilized to detect the expression level of PD-L1. We also evaluated the chemotaxis ability of neutrophils using a Transwell system, assessed the viability and apoptosis rate of GC cells using CCK-8 and flow cytometry, measured the proportions of CD8+PD-1+ and CD8+GZMB+ cells, determined the expression levels of IL-6, TNFα, IFN-γ, IL-8, IL-12, IL-1β, and GZMB by utilizing enzyme-linked immunosorbent assay (ELISA), and examined the expression levels of P2RX1 and PD-L1 using western blot (WB). By establishing a xenograft mouse model, we studied the impact of P2RX1-blocked neutrophils on the efficacy of ICI treatment in the GC microenvironment. RESULTS In GC, clinical analysis revealed increased infiltration of P2RX1-lowly expressed neutrophil subsets and increased expression of PD-L1. In vitro experiments demonstrated that abnormal expression of P2RX1 affected neutrophil function. Furthermore, the blockage or knockdown of P2RX1 in neutrophils modulated CD8+ T cell function, promoting GC progression. In in vivo experiments, the blockage of P2RX1 in neutrophils inhibited the effectiveness of ICI treatment in the GC microenvironment. CONCLUSION This project validated that the loss of P2RX1 in neutrophils induces CD8+ T cell dysfunction and affects the GC development, indicating that P2RX1 may be an accurate biomarker for predicting ICI response, thus providing a theoretical basis for the clinical application of ICI.
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Affiliation(s)
- Yan Zhang
- Department of Medical Oncology, Ma'anshan People's Hospital, 519 Hunan East Road, Huashan District, Ma'anshan City, Anhui Province 243000, China.
| | - Fenglin Zhang
- Department of Medical Oncology, Ma'anshan People's Hospital, 519 Hunan East Road, Huashan District, Ma'anshan City, Anhui Province 243000, China
| | - Zhi Liu
- Department of Pathology, Ma'anshan People's Hospital, 519 Hunan East Road, Huashan District, Ma'anshan City, Anhui Province 243000, China
| | - Min Li
- Department of Medical Oncology, Ma'anshan People's Hospital, 519 Hunan East Road, Huashan District, Ma'anshan City, Anhui Province 243000, China
| | - Ge Wu
- Department of Medical Oncology, Ma'anshan People's Hospital, 519 Hunan East Road, Huashan District, Ma'anshan City, Anhui Province 243000, China
| | - Hui Li
- Department of Medical Oncology, Ma'anshan People's Hospital, 519 Hunan East Road, Huashan District, Ma'anshan City, Anhui Province 243000, China
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Matera MG, Calzetta L, Rinaldi B, Belardo C, Facciolo F, Gallina FT, Page CP, Cazzola M, Rogliani P. Interaction between fluticasone furoate and umeclidinium in passively sensitized isolated human airways. Pulm Pharmacol Ther 2024; 87:102331. [PMID: 39393521 DOI: 10.1016/j.pupt.2024.102331] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/12/2024] [Revised: 09/16/2024] [Accepted: 10/08/2024] [Indexed: 10/13/2024]
Abstract
Asthma management often includes inhaled corticosteroids (ICSs), with additional controllers like long-acting muscarinic antagonists (LAMAs) for severe cases. The primary goal of this study was to investigate the pharmacological interaction between various concentrations of fluticasone furoate (FF) and umeclidinium (UME) in isolated human airways to determine the nature of their interaction, whether synergistic or additive. Medium bronchi and small airways obtained from patients undergoing lobectomy were passively sensitized to mimic asthmatic conditions. The effects of FF and UME, alone and in combination, on airway relaxation were evaluated using histamine-induced contraction and electrical field stimulation. Pharmacological interactions were analyzed using the Bliss Independence theory. Results indicated that FF induced a partial, concentration-dependent relaxation of sensitized airways, while UME induced a larger relaxation in medium bronchi but a weaker effect in small airways. The combination of FF and UME resulted in significantly greater relaxation than either drug alone, demonstrating synergism at high concentrations in medium bronchi but only additive effects in small airways. This study suggests that higher doses of FF might be necessary in a fixed dose combination to achieve optimal synergistic bronchodilation with UME. Future research should focus on clinical trials to confirm these findings and explore the molecular mechanisms underlying these interactions, potentially improving personalized asthma therapy.
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Affiliation(s)
- Maria Gabriella Matera
- Unit of Pharmacology, Department of Experimental Medicine, University of Campania "Luigi Vanvitelli", Naples, Italy
| | - Luigino Calzetta
- Respiratory Disease and Lung Function Unit, Department of Medicine and Surgery, University of Parma, Parma, Italy.
| | - Barbara Rinaldi
- Unit of Pharmacology, Department of Experimental Medicine, University of Campania "Luigi Vanvitelli", Naples, Italy
| | - Carmela Belardo
- Unit of Pharmacology, Department of Experimental Medicine, University of Campania "Luigi Vanvitelli", Naples, Italy
| | - Francesco Facciolo
- Thoracic Surgery Unit, IRCCS Regina Elena National Cancer Institute, Rome, Italy
| | | | - Clive P Page
- Institute of Pharmaceutical Science, King's College London, London, UK
| | - Mario Cazzola
- Unit of Respiratory Medicine, Department of Experimental Medicine, University of Rome "Tor Vergata", Rome, Italy
| | - Paola Rogliani
- Unit of Respiratory Medicine, Department of Experimental Medicine, University of Rome "Tor Vergata", Rome, Italy
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Lim XR, Mercer M, Harraz OF, Hollywood MA, Sergeant GP, Thornbury KD. Evidence of an excitatory purinergic innervation in mouse corpus cavernosum smooth muscle. J Sex Med 2024; 21:977-985. [PMID: 39234981 DOI: 10.1093/jsxmed/qdae107] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/08/2023] [Revised: 06/14/2024] [Accepted: 08/14/2024] [Indexed: 09/06/2024]
Abstract
BACKGROUND Evidence suggests that the corpus cavernosum smooth muscle (CCSM) cells of several species, including humans, express purinergic P2X receptors, but it is not known if the corpus cavernosum has an excitatory purinergic innervation. AIM In this study we aimed to determine if the mouse CCSM has a functional purinergic innervation. METHODS Mouse CCSM myocytes were enzymatically isolated and studied using the perforated patch configuration of the patch clamp technique. Isometric tension was measured in whole cavernosum tissue subjected to electrical field stimulation (EFS) to evoke nerve-mediated responses. OUTCOMES The mouse CCSM myocytes expressed P2X1 receptors, and adenosine triphosphate (ATP) evoked inward currents in these cells. In addition, P2X1-mediated contractions were recorded in whole tissue in response to EFS. RESULTS In cells held under a voltage clamp at -60 mV, ATP (1 μm) evoked large inward currents (mean approximately 900 pA). This current rapidly declined but was repeatable at 8-minute intervals. α,β-methylene ATP (10 μM), an agonist of P2X1 and P2X3 receptors, caused a similar current that also rapidly declined. Desensitization to α,β-methylene ATP negated the effect of ATP, but the ATP effect was restored 8 minutes after washout of α,β-methylene ATP. The effect of ATP was reversibly blocked by NF449 (1 μm), a selective antagonist of P2X1 receptors. In isometric tension experiments electrical field stimulation (EFS) at 0.5-8 Hz evoked frequency-dependent contractions in the presence of l-nitro arginine (l-NO-Arg) (100 μm). When phentolamine (3 μm) and atropine (1 μm) were applied, there remained a nonadrenergic, noncholinergic component of the response to EFS, consisting mainly of a transient contraction. This was significantly reduced by NF449 (1 μm). Finally, in immunocytochemistry experiments, isolated CCSM myocytes stained positively when exposed to an antibody raised against P2X1 receptors. CLINICAL IMPLICATIONS Previous studies have shown that P2X1 receptors in CCSM are upregulated in diabetes. These findings, taken together with the functional evidence presented here, indicate that P2X1 receptors may provide an alternative therapeutic target for treatment of erectile dysfunction in patients with diabetes, which is known to be relatively resistant to treatment with phosphodiesterase 5 inhibitors. STRENGTHS AND LIMITATIONS Strengths of this study are the use of a combination of functional experiments (patch clamp) and immunocytochemical analyses to show expression of P2X1 receptors on CCSM myocytes while also performing functional experiments to show that stimulation these receptors results in contraction of CCSM. A limitation of this study was the use of animal rather than human tissue. CONCLUSION This investigation provides evidence that mouse corpus cavernosum smooth muscle cells express P2X1 receptors and that these receptors are involved in mediating part of the contractile response to nerve stimulation evoked by EFS.
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Affiliation(s)
- Xin Rui Lim
- Department of Pharmacology, Larner College of Medicine, University of Vermont, Burlington VT 05405, United States
| | - Mitchell Mercer
- Smooth Muscle Research Centre, Dundalk Institute of Technology, Dundalk, County Louth A91 K584, Ireland
| | - Osama F Harraz
- Department of Pharmacology, Larner College of Medicine, University of Vermont, Burlington VT 05405, United States
| | - Mark A Hollywood
- Smooth Muscle Research Centre, Dundalk Institute of Technology, Dundalk, County Louth A91 K584, Ireland
| | - Gerard P Sergeant
- Smooth Muscle Research Centre, Dundalk Institute of Technology, Dundalk, County Louth A91 K584, Ireland
| | - Keith D Thornbury
- Smooth Muscle Research Centre, Dundalk Institute of Technology, Dundalk, County Louth A91 K584, Ireland
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Liu L, Bulla LA. Cell death signaling in Anopheles gambiae initiated by Bacillus thuringiensis Cry4B toxin involves Na +/K + ATPase. Exp Biol Med (Maywood) 2023; 248:1191-1205. [PMID: 37642306 PMCID: PMC10621475 DOI: 10.1177/15353702231188072] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/01/2023] [Accepted: 06/10/2023] [Indexed: 08/31/2023] Open
Abstract
Identifying the mechanisms by which bacterial pathogens kill host cells is fundamental to understanding how to control and prevent human and animal disease. In the case of Bacillus thuringiensis (Bt), such knowledge is critical to using the bacterium to kill insect vectors that transmit human and animal disease. For the Cry4B toxin produced by Bt, its capacity to kill Anopheles gambiae, the primary mosquito vector of malaria, is the consequence of a variety of signaling activities. We show here that Cry4B, acting as first messenger, binds specifically to the bitopic cadherin BT-R3 G-protein-coupled receptor (GPCR) localized in the midgut of A. gambiae, activating the downstream second messenger cyclic adenosine monophosphate (cAMP). The direct result of the Cry4B-BT-R3 binding is the release of αs from the heterotrimeric αβγ-G-protein complex and its activation of adenylyl cyclase (AC). The upshot is an increased level of cAMP, which activates protein kinase A (PKA). The functional impact of cAMP-PKA signaling is the stimulation of Na+/K+-ATPase (NKA) which serves as an Na+/K+ pump to maintain proper gradients of extracellular Na+ and intracellular K+. Increased level of cAMP amplifies NKA and upsets normal ion concentration gradients. NKA, as a scaffolding protein, accelerates the first messenger signal to the nucleus, generating additional BT-R3 molecules and promoting their exocytotic trafficking to the cell membrane. Accumulation of BT-R3 on the cell surface facilitates recruitment of additional toxin molecules which, in turn, amplify the original signal in a cascade-like manner. This report provides the first evidence of a bacterial toxin using NKA via AC/PKA signaling to execute cell death.
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Affiliation(s)
- Li Liu
- Department of Biological Sciences, The University of Texas at Dallas, Richardson, TX 75080-3021 USA
| | - Lee A Bulla
- Department of Biological Sciences, The University of Texas at Dallas, Richardson, TX 75080-3021 USA
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Abstract
Within the family of purinergic receptors, the P2X1 receptor is a ligand-gated ion channel that plays a role in urogenital, immune and cardiovascular function. Specifically, the P2X1 receptor has been implicated in controlling smooth muscle contractions of the vas deferens and therefore has emerged as an exciting drug target for male contraception. In addition, the P2X1 receptor contributes to smooth muscle contractions of the bladder and is a target to treat bladder dysfunction. Finally, platelets and neutrophils have populations of P2X1 receptors that could be targeted for thrombosis and inflammatory conditions. Drugs that specifically target the P2X1 receptor have been challenging to develop, and only recently have small molecule antagonists of the P2X1 receptor been available. However, these ligands need further biological validation for appropriate selectivity and drug-like properties before they will be suitable for use in preclinical models of disease. Although the atomic structure of the P2X1 receptor has yet to be determined, the recent discovery of several other P2X receptor structures and improvements in the field of structural biology suggests that this is now a distinct possibility. Such efforts may significantly improve drug discovery efforts at the P2X1 receptor.
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Mittal A, Park PD, Mitchell R, Fang H, Bagher P. Comparison of Adrenergic and Purinergic Receptor Contributions to Vasomotor Responses in Mesenteric Arteries of C57BL/6J Mice and Wistar Rats. J Vasc Res 2020; 58:1-15. [PMID: 33311016 DOI: 10.1159/000511462] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/16/2020] [Accepted: 09/07/2020] [Indexed: 11/19/2022] Open
Abstract
INTRODUCTION The sympathetic nervous system can modulate arteriolar tone through release of adenosine triphosphate and norepinephrine, which bind to purinergic and adrenergic receptors (ARs), respectively. The expression pattern of these receptors, as well as the composition of neurotransmitters released from perivascular nerves (PVNs), can vary both in organ systems within and across species, such as mice and rats. OBJECTIVE This study explores the function of α1A subtypes in mouse and rat third-order mesenteric arteries and investigates PVN-mediated vasoconstriction to identify which neurotransmitters are released from sympathetic PVNs. METHODS Third-order mesenteric arteries from male C57BL/6J mice and Wistar rats were isolated and mounted on a wire myograph for functional assessment. Arteries were exposed to phenylephrine (PE) and then incubated with either α1A antagonist RS100329 (RS) or α1D antagonist BMY7378, before reexposure to PE. Electrical field stimulation was performed by passing current through platinum electrodes positioned adjacent to arteries in the absence and presence of a nonspecific alpha AR blocker phentolamine and/or P2X1-specific purinergic receptor blocker NF449. RESULTS Inhibition of α1 ARs by RS revealed that PE-induced vasoconstriction is primarily mediated through α1A and that the contribution of the α1A AR is greater in rats than in mice. In the mouse model, sympathetic nerve-mediated vasoconstriction is mediated by both ARs and purinergic receptors, whereas in rats, vasoconstriction appeared to only be mediated by ARs and a nonpurinergic neurotransmitter. Further, neither model demonstrated that α1D ARs play a significant role in PE-mediated vasoconstriction. CONCLUSIONS The mesenteric arteries of male C57BL/6J mice and Wistar rats have subtle differences in the signaling mechanisms used to mediate vasoconstriction. As signaling pathways in humans under physiological and pathophysiological conditions become better defined, the current study may inform animal model selection for preclinical studies.
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Affiliation(s)
- Astha Mittal
- Department of Medical Physiology, Texas A&M University Health Science Center, Bryan, Texas, USA
| | - Peter D Park
- Department of Medical Physiology, Texas A&M University Health Science Center, Bryan, Texas, USA
| | - Ray Mitchell
- Department of Medical Physiology, Texas A&M University Health Science Center, Bryan, Texas, USA
| | - Hanwei Fang
- Department of Microbiology and Molecular Medicine University of Geneva, Geneva, Switzerland
| | - Pooneh Bagher
- Department of Medical Physiology, Texas A&M University Health Science Center, Bryan, Texas, USA,
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Tian M, Abdelrahman A, Baqi Y, Fuentes E, Azazna D, Spanier C, Densborn S, Hinz S, Schmid R, Müller CE. Discovery and Structure Relationships of Salicylanilide Derivatives as Potent, Non-acidic P2X1 Receptor Antagonists. J Med Chem 2020; 63:6164-6178. [PMID: 32345019 DOI: 10.1021/acs.jmedchem.0c00435] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Abstract
Antagonists for the ATP-gated ion channel receptor P2X1 have potential as antithrombotics and for treating hyperactive bladder and inflammation. In this study, salicylanilide derivatives were synthesized based on a screening hit. P2X1 antagonistic potency was assessed in 1321N1 astrocytoma cells stably transfected with the human P2X1 receptor by measuring inhibition of the ATP-induced calcium influx. Structure-activity relationships were analyzed, and selectivity versus other P2X receptor subtypes was assessed. The most potent compounds, N-[3,5-bis(trifluoromethyl)phenyl]-5-chloro-2-hydroxybenzamide (1, IC50 0.0192 μM) and N-[3,5-bis(trifluoromethyl)phenyl]-4-chloro-2-hydroxybenzamide (14, IC50 0.0231 μM), displayed >500-fold selectivity versus P2X2 and P2X3, and 10-fold selectivity versus P2X4 and P2X7 receptors, and inhibited collagen-induced platelet aggregation. They behaved as negative allosteric modulators, and molecular modeling studies suggested an extracellular binding site. Besides selective P2X1 antagonists, compounds with ancillary P2X4 and/or P2X7 receptor inhibition were discovered. These compounds represent the first potent, non-acidic, allosteric P2X1 receptor antagonists reported to date.
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Affiliation(s)
- Maoqun Tian
- PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical & Medicinal Chemistry, University of Bonn, An der Immenburg 4, D-53121 Bonn, Germany
| | - Aliaa Abdelrahman
- PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical & Medicinal Chemistry, University of Bonn, An der Immenburg 4, D-53121 Bonn, Germany
| | - Younis Baqi
- Department of Chemistry, Faculty of Science, Sultan Qaboos University, P.O. Box 36, 123 Muscat, Oman
| | - Eduardo Fuentes
- Thrombosis Research Center, Medical Technology School, Department of Clinical Biochemistry and Immunohaematology, Faculty of Health Sciences, Interdisciplinary Center on Aging, Universidad de Talca, 3460000 Talca, Chile
| | - Djamil Azazna
- PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical & Medicinal Chemistry, University of Bonn, An der Immenburg 4, D-53121 Bonn, Germany
| | - Claudia Spanier
- PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical & Medicinal Chemistry, University of Bonn, An der Immenburg 4, D-53121 Bonn, Germany
| | - Sabrina Densborn
- PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical & Medicinal Chemistry, University of Bonn, An der Immenburg 4, D-53121 Bonn, Germany
| | - Sonja Hinz
- PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical & Medicinal Chemistry, University of Bonn, An der Immenburg 4, D-53121 Bonn, Germany
| | - Ralf Schmid
- Department of Molecular and Cell Biology, University of Leicester, Leicester LE1 7RH, U.K.,Leicester Institute of Structural and Chemical Biology, University of Leicester, Leicester LE1 7RH, U.K
| | - Christa E Müller
- PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical & Medicinal Chemistry, University of Bonn, An der Immenburg 4, D-53121 Bonn, Germany
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Mazeh AC, Angus JA, Wright CE. The effects of varying Mg 2+ ion concentrations on contractions to the cotransmitters ATP and noradrenaline in the rat vas deferens. Auton Neurosci 2019; 222:102588. [PMID: 31669796 DOI: 10.1016/j.autneu.2019.102588] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/19/2019] [Revised: 08/30/2019] [Accepted: 09/13/2019] [Indexed: 10/25/2022]
Abstract
The vas deferens responds to a single electrical pulse with a biphasic contraction caused by cotransmitters ATP and noradrenaline. Removing Mg2+ (normally 1.2 mM) from the physiological salt solution (PSS) enhances the contraction. This study aimed to determine the effect of Mg2+ concentration on nerve cotransmitter-mediated contractions. Rat vasa deferentia were sequentially bathed in increasing (0, 1.2, 3 mM) or decreasing (3, 1.2, 0 mM) Mg2+ concentrations. At each concentration a single field pulse was applied, and the biphasic contraction recorded. Contractions to exogenous noradrenaline 10 μM and ATP 100 μM were also determined. The biphasic nerve-mediated contraction was elicited by ATP and noradrenaline as NF449 (10 μM) and prazosin (100 nM) completely prevented the respective peaks. Taking the contractions in normal PSS (Mg2+ 1.2 mM) as 100%, lowering Mg2+ to 0 mM enhanced the ATP peak to 170 ± 7% and raising Mg2+ to 3 mM decreased it to 39 ± 3%; the noradrenaline peak was not affected by lowering Mg2+ to 0 mM (97 ± 3%) but was decreased to 63 ± 4% in high Mg2+ (3 mM). Contractions to exogenous ATP, but not noradrenaline, were increased in Mg2+ 0 mM and both were inhibited with Mg2+ 3 mM. Changing Mg2+ concentration affects the contractions elicited by the cotransmitters ATP and noradrenaline. The greatest effects were to potentiate the contraction to ATP in Mg2+ 0 mM and to inhibit the contraction to both ATP and noradrenaline in high Mg2+. Future publications should clearly justify any decision to vary the magnesium concentration from normal (1.2 mM) values.
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Affiliation(s)
- Amna C Mazeh
- Cardiovascular Therapeutics Unit, Department of Pharmacology and Therapeutics, University of Melbourne, Victoria 3010, Australia.
| | - James A Angus
- Cardiovascular Therapeutics Unit, Department of Pharmacology and Therapeutics, University of Melbourne, Victoria 3010, Australia.
| | - Christine E Wright
- Cardiovascular Therapeutics Unit, Department of Pharmacology and Therapeutics, University of Melbourne, Victoria 3010, Australia.
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Identification of aurintricarboxylic acid as a potent allosteric antagonist of P2X1 and P2X3 receptors. Neuropharmacology 2019; 158:107749. [PMID: 31461640 DOI: 10.1016/j.neuropharm.2019.107749] [Citation(s) in RCA: 40] [Impact Index Per Article: 6.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/03/2019] [Revised: 08/14/2019] [Accepted: 08/23/2019] [Indexed: 12/13/2022]
Abstract
The homotrimeric P2X3 receptor, one of the seven members of the ATP-gated P2X receptor family, plays a crucial role in sensory neurotransmission. P2X3 receptor antagonists have been identified as promising drugs to treat chronic cough and are suggested to offer pain relief in chronic pain such as neuropathic pain. Here, we analysed whether compounds affect P2X3 receptor activity by high-throughput screening of the Spectrum Collection of 2000 approved drugs, natural products and bioactive substances. We identified aurintricarboxylic acid (ATA) as a nanomolar-potency antagonist of P2X3 receptor-mediated responses. Two-electrode voltage clamp electrophysiology-based concentration-response analysis and selectivity profiling revealed that ATA strongly inhibits the rP2X1 and rP2X3 receptors (with IC50 values of 8.6 nM and 72.9 nM, respectively) and more weakly inhibits P2X2/3, P2X2, P2X4 or P2X7 receptors (IC50 values of 0.76 μM, 22 μM, 763 μM or 118 μM, respectively). Patch-clamp analysis of mouse DRG neurons revealed that ATA inhibited native P2X3 and P2X2/3 receptors to a similar extent than rat P2X3 and P2X2/3 receptors expressed in Xenopus oocytes. In a radioligand binding assay, up to 30 μM ATA did not compete with [3H]-ATP for rP2X3 receptor binding, indicating a non-competitive mechanism of action. Molecular docking studies, site-directed mutagenesis and concentration-response analysis revealed that ATA binds to the negative allosteric site of the hP2X3 receptor. In summary, ATA as a drug-like pharmacological tool compound is a nanomolar-potency, allosteric antagonist with selectivity towards αβ-methylene-ATP-sensitive P2X1 and P2X3 receptors.
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The ATP-gated P2X 1 ion channel contributes to the severity of antibody-mediated Transfusion-Related Acute Lung Injury in mice. Sci Rep 2019; 9:5159. [PMID: 30914724 PMCID: PMC6435740 DOI: 10.1038/s41598-019-41742-9] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/18/2018] [Accepted: 03/11/2019] [Indexed: 12/19/2022] Open
Abstract
The biological responses that control the development of Transfusion-Related Acute Lung Injury (TRALI), a serious post-transfusion respiratory syndrome, still need to be clarified. Since extracellular nucleotides and their P2 receptors participate in inflammatory processes as well as in cellular responses to stress, we investigated the role of the ATP-gated P2X1 cation channel in antibody-mediated TRALI. The effects of NF449, a selective P2X1 receptor (P2RX1) antagonist, were analyzed in a mouse two-hit model of TRALI. Mice were primed with lipopolysaccharide (LPS) and 24 h later challenged by administrating an anti-MHC I antibody. The selective P2RX1 antagonist NF449 was administrated before the administration of LPS and/or the anti-MHC I antibody. When given before antibody administration, NF449 improved survival while maximal protection was achieved when NF449 was also administrated before the sensitization step. Under this later condition, protein contents in bronchoalveolar lavages were dramatically reduced. Cell depletion experiments indicated that monocytes/macrophages, but not neutrophils, contribute to this effect. In addition, the reduced lung periarteriolar interstitial edemas in NF449-treated mice suggested that P2RX1 from arteriolar smooth muscle cells could represent a target of NF449. Accordingly, inhibition of TRPC6, another cation channel expressed by smooth muscle cells, also reduced TRALI-associated pulmonary interstitial and alveolar edemas. These data strongly suggest that cation channels like P2RX1 or TRPC6 participate to TRALI pathological responses.
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Jung YH, Kim YO, Lin H, Cho JH, Park JH, Lee SD, Bae J, Kang KM, Kim YG, Pae AN, Ko H, Park CS, Yoon MH, Kim YC. Discovery of Potent Antiallodynic Agents for Neuropathic Pain Targeting P2X3 Receptors. ACS Chem Neurosci 2017; 8:1465-1478. [PMID: 28323403 DOI: 10.1021/acschemneuro.6b00401] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022] Open
Abstract
Antagonism of the P2X3 receptor is one of the potential therapeutic strategies for the management of neuropathic pain because P2X3 receptors are predominantly localized on small to medium diameter C- and Aδ-fiber primary afferent neurons, which are related to the pain-sensing system. In this study, 5-hydroxy pyridine derivatives were designed, synthesized, and evaluated for their in vitro biological activities by two-electrode voltage clamp assay at hP2X3 receptors. Among the novel hP2X3 receptor antagonists, intrathecal treatment of compound 29 showed parallel efficacy with pregabalin (calcium channel modulator) and higher efficacy than AF353 (P2X3 receptor antagonist) in the evaluation of its antiallodynic effects in spinal nerve ligation rats. However, because compound 29 was inactive by intraperitoneal administration in neuropathic pain animal models due to low cell permeability, the corresponding methyl ester analogue, 28, which could be converted to compound 29 in vivo, was investigated as a prodrug concept. Intravenous injection of compound 28 resulted in potent antiallodynic effects, with ED50 values of 2.62 and 2.93 mg/kg in spinal nerve ligation and chemotherapy-induced peripheral neuropathy rats, respectively, indicating that new drug development targeting the P2X3 receptor could be promising for neuropathic pain, a disease with high unmet medical needs.
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Affiliation(s)
- Young-Hwan Jung
- School of Life Sciences, Gwangju Institute of Science and Technology (GIST), Gwangju 500-712, Republic of Korea
| | | | | | - Joong-Heui Cho
- New Drug Development Center
(NDDC), Daegu-Gyeongbuk Medical Innovation Foundation (DGMIF), 80
Cheombok-ro, Dong-gu, Daegu 41061, Republic of Korea
| | - Jin-Hee Park
- New Drug Development Center
(NDDC), Daegu-Gyeongbuk Medical Innovation Foundation (DGMIF), 80
Cheombok-ro, Dong-gu, Daegu 41061, Republic of Korea
| | - So-Deok Lee
- School of Life Sciences, Gwangju Institute of Science and Technology (GIST), Gwangju 500-712, Republic of Korea
| | - Jinsu Bae
- Department of Biomedical Science and Engineering, Gwangju Institute of Science and Technology (GIST), Gwangju 500-712, Republic of Korea
| | - Koon Mook Kang
- School of Life Sciences, Gwangju Institute of Science and Technology (GIST), Gwangju 500-712, Republic of Korea
| | - Yoon-Gyoon Kim
- College of Pharmacy, Dankook University, Cheonan 330-714, Republic of Korea
| | - Ae Nim Pae
- Convergence Research Center for Diagnosis, Treatment
and Care System of Dementia, Korea Institute of Science and Technology, PO Box 131, Cheongryang, Seoul 130-650, Republic of Korea
| | - Hyojin Ko
- School of Life Sciences, Gwangju Institute of Science and Technology (GIST), Gwangju 500-712, Republic of Korea
| | - Chul-Seung Park
- School of Life Sciences, Gwangju Institute of Science and Technology (GIST), Gwangju 500-712, Republic of Korea
| | | | - Yong-Chul Kim
- School of Life Sciences, Gwangju Institute of Science and Technology (GIST), Gwangju 500-712, Republic of Korea
- Department of Biomedical Science and Engineering, Gwangju Institute of Science and Technology (GIST), Gwangju 500-712, Republic of Korea
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13
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Stallaert W, van der Westhuizen ET, Schönegge AM, Plouffe B, Hogue M, Lukashova V, Inoue A, Ishida S, Aoki J, Le Gouill C, Bouvier M. Purinergic Receptor Transactivation by the β2-Adrenergic Receptor Increases Intracellular Ca 2+ in Nonexcitable Cells. Mol Pharmacol 2017; 91:533-544. [PMID: 28280061 DOI: 10.1124/mol.116.106419] [Citation(s) in RCA: 55] [Impact Index Per Article: 6.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/14/2016] [Accepted: 03/06/2017] [Indexed: 12/11/2022] Open
Abstract
The β2 adrenergic receptor (β2AR) increases intracellular Ca2+ in a variety of cell types. By combining pharmacological and genetic manipulations, we reveal a novel mechanism through which the β2AR promotes Ca2+ mobilization (pEC50 = 7.32 ± 0.10) in nonexcitable human embryonic kidney (HEK)293S cells. Downregulation of Gs with sustained cholera toxin pretreatment and the use of Gs-null HEK293 (∆Gs-HEK293) cells generated using the clustered regularly interspaced short palindromic repeat-associated protein-9 nuclease (CRISPR/Cas9) system, combined with pharmacological modulation of cAMP formation, revealed a Gs-dependent but cAMP-independent increase in intracellular Ca2+ following β2AR stimulation. The increase in cytoplasmic Ca2+ was inhibited by P2Y purinergic receptor antagonists as well as a dominant-negative mutant form of Gq, a Gq-selective inhibitor, and an inositol 1,4,5-trisphosphate (IP3) receptor antagonist, suggesting a role for this Gq-coupled receptor family downstream of the β2AR activation. Consistent with this mechanism, β2AR stimulation promoted the extracellular release of ATP, and pretreatment with apyrase inhibited the β2AR-promoted Ca2+ mobilization. Together, these data support a model whereby the β2AR stimulates a Gs-dependent release of ATP, which transactivates Gq-coupled P2Y receptors through an inside-out mechanism, leading to a Gq- and IP3-dependent Ca2+ mobilization from intracellular stores. Given that β2AR and P2Y receptors are coexpressed in various tissues, this novel signaling paradigm could be physiologically important and have therapeutic implications. In addition, this study reports the generation and validation of HEK293 cells deleted of Gs using the CRISPR/Cas9 genome editing technology that will undoubtedly be powerful tools to study Gs-dependent signaling.
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Affiliation(s)
- Wayne Stallaert
- Department of Biochemistry (W.S., E.T.v.d.W., A.-M.S., B.P., M.B.) and Institute for Research in Immunology and Cancer (W.S., E.T.v.d.W., A.-M.S., B.P., M.H., V.L., C.L.G., M.B.), Université de Montréal, Montréal, QC, Canada; Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Miyagi, Japan (A.I., S.I., J.A.); Japan Science and Technology Agency, Precursory Research for Embryonic Science and Technology, Kawaguchi, Saitama, Japan (A.I.); and Japan Agency for Medical Research and Development, Core Research for Evolutional Science and Technology, Chiyoda-ku, Tokyo, Japan (J.A.)
| | - Emma T van der Westhuizen
- Department of Biochemistry (W.S., E.T.v.d.W., A.-M.S., B.P., M.B.) and Institute for Research in Immunology and Cancer (W.S., E.T.v.d.W., A.-M.S., B.P., M.H., V.L., C.L.G., M.B.), Université de Montréal, Montréal, QC, Canada; Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Miyagi, Japan (A.I., S.I., J.A.); Japan Science and Technology Agency, Precursory Research for Embryonic Science and Technology, Kawaguchi, Saitama, Japan (A.I.); and Japan Agency for Medical Research and Development, Core Research for Evolutional Science and Technology, Chiyoda-ku, Tokyo, Japan (J.A.)
| | - Anne-Marie Schönegge
- Department of Biochemistry (W.S., E.T.v.d.W., A.-M.S., B.P., M.B.) and Institute for Research in Immunology and Cancer (W.S., E.T.v.d.W., A.-M.S., B.P., M.H., V.L., C.L.G., M.B.), Université de Montréal, Montréal, QC, Canada; Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Miyagi, Japan (A.I., S.I., J.A.); Japan Science and Technology Agency, Precursory Research for Embryonic Science and Technology, Kawaguchi, Saitama, Japan (A.I.); and Japan Agency for Medical Research and Development, Core Research for Evolutional Science and Technology, Chiyoda-ku, Tokyo, Japan (J.A.)
| | - Bianca Plouffe
- Department of Biochemistry (W.S., E.T.v.d.W., A.-M.S., B.P., M.B.) and Institute for Research in Immunology and Cancer (W.S., E.T.v.d.W., A.-M.S., B.P., M.H., V.L., C.L.G., M.B.), Université de Montréal, Montréal, QC, Canada; Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Miyagi, Japan (A.I., S.I., J.A.); Japan Science and Technology Agency, Precursory Research for Embryonic Science and Technology, Kawaguchi, Saitama, Japan (A.I.); and Japan Agency for Medical Research and Development, Core Research for Evolutional Science and Technology, Chiyoda-ku, Tokyo, Japan (J.A.)
| | - Mireille Hogue
- Department of Biochemistry (W.S., E.T.v.d.W., A.-M.S., B.P., M.B.) and Institute for Research in Immunology and Cancer (W.S., E.T.v.d.W., A.-M.S., B.P., M.H., V.L., C.L.G., M.B.), Université de Montréal, Montréal, QC, Canada; Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Miyagi, Japan (A.I., S.I., J.A.); Japan Science and Technology Agency, Precursory Research for Embryonic Science and Technology, Kawaguchi, Saitama, Japan (A.I.); and Japan Agency for Medical Research and Development, Core Research for Evolutional Science and Technology, Chiyoda-ku, Tokyo, Japan (J.A.)
| | - Viktoria Lukashova
- Department of Biochemistry (W.S., E.T.v.d.W., A.-M.S., B.P., M.B.) and Institute for Research in Immunology and Cancer (W.S., E.T.v.d.W., A.-M.S., B.P., M.H., V.L., C.L.G., M.B.), Université de Montréal, Montréal, QC, Canada; Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Miyagi, Japan (A.I., S.I., J.A.); Japan Science and Technology Agency, Precursory Research for Embryonic Science and Technology, Kawaguchi, Saitama, Japan (A.I.); and Japan Agency for Medical Research and Development, Core Research for Evolutional Science and Technology, Chiyoda-ku, Tokyo, Japan (J.A.)
| | - Asuka Inoue
- Department of Biochemistry (W.S., E.T.v.d.W., A.-M.S., B.P., M.B.) and Institute for Research in Immunology and Cancer (W.S., E.T.v.d.W., A.-M.S., B.P., M.H., V.L., C.L.G., M.B.), Université de Montréal, Montréal, QC, Canada; Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Miyagi, Japan (A.I., S.I., J.A.); Japan Science and Technology Agency, Precursory Research for Embryonic Science and Technology, Kawaguchi, Saitama, Japan (A.I.); and Japan Agency for Medical Research and Development, Core Research for Evolutional Science and Technology, Chiyoda-ku, Tokyo, Japan (J.A.)
| | - Satoru Ishida
- Department of Biochemistry (W.S., E.T.v.d.W., A.-M.S., B.P., M.B.) and Institute for Research in Immunology and Cancer (W.S., E.T.v.d.W., A.-M.S., B.P., M.H., V.L., C.L.G., M.B.), Université de Montréal, Montréal, QC, Canada; Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Miyagi, Japan (A.I., S.I., J.A.); Japan Science and Technology Agency, Precursory Research for Embryonic Science and Technology, Kawaguchi, Saitama, Japan (A.I.); and Japan Agency for Medical Research and Development, Core Research for Evolutional Science and Technology, Chiyoda-ku, Tokyo, Japan (J.A.)
| | - Junken Aoki
- Department of Biochemistry (W.S., E.T.v.d.W., A.-M.S., B.P., M.B.) and Institute for Research in Immunology and Cancer (W.S., E.T.v.d.W., A.-M.S., B.P., M.H., V.L., C.L.G., M.B.), Université de Montréal, Montréal, QC, Canada; Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Miyagi, Japan (A.I., S.I., J.A.); Japan Science and Technology Agency, Precursory Research for Embryonic Science and Technology, Kawaguchi, Saitama, Japan (A.I.); and Japan Agency for Medical Research and Development, Core Research for Evolutional Science and Technology, Chiyoda-ku, Tokyo, Japan (J.A.)
| | - Christian Le Gouill
- Department of Biochemistry (W.S., E.T.v.d.W., A.-M.S., B.P., M.B.) and Institute for Research in Immunology and Cancer (W.S., E.T.v.d.W., A.-M.S., B.P., M.H., V.L., C.L.G., M.B.), Université de Montréal, Montréal, QC, Canada; Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Miyagi, Japan (A.I., S.I., J.A.); Japan Science and Technology Agency, Precursory Research for Embryonic Science and Technology, Kawaguchi, Saitama, Japan (A.I.); and Japan Agency for Medical Research and Development, Core Research for Evolutional Science and Technology, Chiyoda-ku, Tokyo, Japan (J.A.)
| | - Michel Bouvier
- Department of Biochemistry (W.S., E.T.v.d.W., A.-M.S., B.P., M.B.) and Institute for Research in Immunology and Cancer (W.S., E.T.v.d.W., A.-M.S., B.P., M.H., V.L., C.L.G., M.B.), Université de Montréal, Montréal, QC, Canada; Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Miyagi, Japan (A.I., S.I., J.A.); Japan Science and Technology Agency, Precursory Research for Embryonic Science and Technology, Kawaguchi, Saitama, Japan (A.I.); and Japan Agency for Medical Research and Development, Core Research for Evolutional Science and Technology, Chiyoda-ku, Tokyo, Japan (J.A.)
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14
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Faulks M, Kuit TA, Sophocleous RA, Curtis BL, Curtis SJ, Jurak LM, Sluyter R. P2X7 receptor activation causes phosphatidylserine exposure in canine erythrocytes. World J Hematol 2016; 5:88-93. [DOI: 10.5315/wjh.v5.i4.88] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/20/2016] [Revised: 08/10/2016] [Accepted: 09/22/2016] [Indexed: 02/05/2023] Open
Abstract
AIM To determine if activation of the ATP-gated P2X7 receptor channel induces phosphatidylserine (PS) exposure in erythrocytes from multiple dog breeds.
METHODS Peripheral blood was collected from 25 dogs representing 13 pedigrees and seven crossbreeds. ATP-induced PS exposure on canine erythrocytes in vitro was assessed using a flow cytometric Annexin V binding assay.
RESULTS ATP induced PS exposure in erythrocytes from all dogs studied. ATP caused PS exposure in a concentration-dependent manner with an EC50 value of 395 μmol/L. The non-P2X7 agonists, ADP or AMP, did not cause PS exposure. The P2X7 antagonist, AZ10606120, but not the P2X1 antagonist, NF449, blocked ATP-induced PS exposure.
CONCLUSION The results indicate that ATP induces PS exposure in erythrocytes from various dog breeds and that this process is mediated by P2X7 activation.
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15
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Gaval-Cruz M, Goertz RB, Puttick DJ, Bowles DE, Meyer RC, Hall RA, Ko D, Paladini CA, Weinshenker D. Chronic loss of noradrenergic tone produces β-arrestin2-mediated cocaine hypersensitivity and alters cellular D2 responses in the nucleus accumbens. Addict Biol 2016; 21:35-48. [PMID: 25123018 DOI: 10.1111/adb.12174] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Abstract
Cocaine blocks plasma membrane monoamine transporters and increases extracellular levels of dopamine (DA), norepinephrine (NE) and serotonin (5-HT). The addictive properties of cocaine are mediated primarily by DA, while NE and 5-HT play modulatory roles. Chronic inhibition of dopamine β-hydroxylase (DBH), which converts DA to NE, increases the aversive effects of cocaine and reduces cocaine use in humans, and produces behavioral hypersensitivity to cocaine and D2 agonism in rodents, but the underlying mechanism is unknown. We found a decrease in β-arrestin2 (βArr2) in the nucleus accumbens (NAc) following chronic genetic or pharmacological DBH inhibition, and overexpression of βArr2 in the NAc normalized cocaine-induced locomotion in DBH knockout (Dbh -/-) mice. The D2/3 agonist quinpirole decreased excitability in NAc medium spiny neurons (MSNs) from control, but not Dbh -/- animals, where instead there was a trend for an excitatory effect. The Gαi inhibitor NF023 abolished the quinpirole-induced decrease in excitability in control MSNs, but had no effect in Dbh -/- MSNs, whereas the Gαs inhibitor NF449 restored the ability of quinpirole to decrease excitability in Dbh -/- MSNs, but had no effect in control MSNs. These results suggest that chronic loss of noradrenergic tone alters behavioral responses to cocaine via decreases in βArr2 and cellular responses to D2/D3 activation, potentially via changes in D2-like receptor G-protein coupling in NAc MSNs.
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Affiliation(s)
- Meriem Gaval-Cruz
- Department of Human Genetics; Emory University School of Medicine; Atlanta GA USA
| | - Richard B. Goertz
- Department of Biology; Neurosciences Institute; University of Texas at San Antonio; San Antonio TX USA
| | - Daniel J. Puttick
- Department of Human Genetics; Emory University School of Medicine; Atlanta GA USA
| | - Dawn E. Bowles
- Department of Surgery; Duke University School of Medicine; Durham NC USA
| | - Rebecca C. Meyer
- Department of Pharmacology; Emory University School of Medicine; Atlanta GA USA
| | - Randy A. Hall
- Department of Pharmacology; Emory University School of Medicine; Atlanta GA USA
| | - Daijin Ko
- Department of Management Science and Statistics; University of Texas at San Antonio; San Antonio TX USA
| | - Carlos A. Paladini
- Department of Biology; Neurosciences Institute; University of Texas at San Antonio; San Antonio TX USA
| | - David Weinshenker
- Department of Human Genetics; Emory University School of Medicine; Atlanta GA USA
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16
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Insights into the channel gating of P2X receptors from structures, dynamics and small molecules. Acta Pharmacol Sin 2016; 37:44-55. [PMID: 26725734 DOI: 10.1038/aps.2015.127] [Citation(s) in RCA: 28] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/15/2015] [Accepted: 11/02/2015] [Indexed: 12/16/2022]
Abstract
P2X receptors, as ATP-gated non-selective trimeric ion channels, are permeable to Na(+), K(+) and Ca(2+). Comparing with other ligand-gated ion channel families, P2X receptors are distinct in their unique gating properties and pathophysiological roles, and have attracted attention as promising drug targets for a variety of diseases, such as neuropathic pain, multiple sclerosis, rheumatoid arthritis and thrombus. Several small molecule inhibitors for distinct P2X subtypes have entered into clinical trials. However, many questions regarding the gating mechanism of P2X remain unsolved. The structural determinations of P2X receptors at the resting and ATP-bound open states revealed that P2X receptor gating is a cooperative allosteric process involving multiple domains, which marks the beginning of the post-structure era of P2X research at atomic level. Here, we review the current knowledge on the structure-function relationship of P2X receptors, depict the whole picture of allosteric changes during the channel gating, and summarize the active sites that may contribute to new strategies for developing novel allosteric drugs targeting P2X receptors.
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17
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Interaction between corticosteroids and muscarinic antagonists in human airways. Pulm Pharmacol Ther 2015; 36:1-9. [PMID: 26656790 DOI: 10.1016/j.pupt.2015.11.004] [Citation(s) in RCA: 49] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/18/2015] [Revised: 11/05/2015] [Accepted: 11/25/2015] [Indexed: 12/30/2022]
Abstract
BACKGROUND To date there is emerging clinical evidence to add long-acting anti-muscarinic agents (LAMAs) with inhaled corticosteroid (ICSs) in asthma, but the pharmacological rationale that supports the use of such a combination has not yet been explained. The aim of this study was to pharmacologically investigate the interaction between the ICS beclomethasone and the LAMA glycopyrronium on the human airway smooth muscle (ASM) tone. METHODS We investigated the rapid non-genomic bronchorelaxant effect of beclomethasone and glycopyrronium, administered alone and in combination, in human isolated bronchi and bronchioles. Experiments were carried out also in passively sensitized airways and the pharmacological analysis of drug interaction was performed by Bliss Independence method. RESULTS The acute administration of beclomethasone and glycopyrronium induced a significant relaxation of passively sensitized ASM pre-contracted with histamine, by causing submaximal/maximal inhibition of the contractile tone in both medium bronchi and bronchioles. Beclomethasone was characterized by a rapid non-genomic and epithelium independent bronchorelaxant effect. In passively sensitized airways, this effect seemed to be dependent by the activation of a Gsα--cyclic adenosine monophosphate (cAMP)--protein kinase A cascade. While no synergistic interaction was detected in non-sensitized bronchi, the beclomethasone/glycopyrronium combination synergistically enhanced the relaxation of passively sensitized medium and small bronchi. The synergistic interaction between beclomethasone and glycopyrronium was associated with an increase of cAMP concentrations. CONCLUSIONS Our study provides for the first time the pharmacological rationale for combining low doses of an ICS plus a LAMA.
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18
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Angus JA, Wright CE. ATP is not involved in α1-adrenoceptor-mediated vasoconstriction in resistance arteries. Eur J Pharmacol 2015; 769:162-6. [PMID: 26593428 DOI: 10.1016/j.ejphar.2015.11.013] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/20/2015] [Revised: 10/12/2015] [Accepted: 11/12/2015] [Indexed: 12/13/2022]
Abstract
Recent publications suggest that α1-adrenoceptor stimulation by exogenous agonists such as phenylephrine in resistance arteries cause contraction through the release of ATP from within the vascular smooth muscle cells. This ATP exits the cell through pannexin-1 channels to act back "autocrine-like" on P2 receptors on the smooth muscle that cause the contraction. In this work we directly test this hypothesis by using a selective P2X1 purinoceptor antagonist NF449 (1-10µM) against phenylephrine and ATP concentration-response curves in small mesenteric arteries of the rat and thoracodorsal arteries of the mouse. We show that NF449 is a simple competitive antagonist of ATP with a pKB of 6.43 and 6.41 in rat and mouse arteries, respectively, but did not antagonise phenylephrine concentration-response curves. This work cautions against the growing overstated role of the reputed pannexin-1/ATP release axis following α1-adrenoceptor activation in small resistance arteries.
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Affiliation(s)
- James A Angus
- Cardiovascular Therapeutics Unit, Department of Pharmacology and Therapeutics, University of Melbourne, Victoria 3010, Australia.
| | - Christine E Wright
- Cardiovascular Therapeutics Unit, Department of Pharmacology and Therapeutics, University of Melbourne, Victoria 3010, Australia.
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19
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Ruepp MD, Brozik JA, de Esch IJP, Farndale RW, Murrell-Lagnado RD, Thompson AJ. A fluorescent approach for identifying P2X1 ligands. Neuropharmacology 2015; 98:13-21. [PMID: 26026951 PMCID: PMC4728187 DOI: 10.1016/j.neuropharm.2015.05.016] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/30/2014] [Revised: 04/02/2015] [Accepted: 05/12/2015] [Indexed: 02/06/2023]
Abstract
There are no commercially available, small, receptor-specific P2X1 ligands. There are several synthetic derivatives of the natural agonist ATP and some structurally-complex antagonists including compounds such as PPADS, NTP-ATP, suramin and its derivatives (e.g. NF279, NF449). NF449 is the most potent and selective ligand, but potencies of many others are not particularly high and they can also act at other P2X, P2Y and non-purinergic receptors. While there is clearly scope for further work on P2X1 receptor pharmacology, screening can be difficult owing to rapid receptor desensitisation. To reduce desensitisation substitutions can be made within the N-terminus of the P2X1 receptor, but these could also affect ligand properties. An alternative is the use of fluorescent voltage-sensitive dyes that respond to membrane potential changes resulting from channel opening. Here we utilised this approach in conjunction with fragment-based drug-discovery. Using a single concentration (300 μM) we identified 46 novel leads from a library of 1443 fragments (hit rate = 3.2%). These hits were independently validated by measuring concentration-dependence with the same voltage-sensitive dye, and by visualising the competition of hits with an Alexa-647-ATP fluorophore using confocal microscopy; confocal yielded kon (1.142 × 106 M−1 s−1) and koff (0.136 s−1) for Alexa-647-ATP (Kd = 119 nM). The identified hit fragments had promising structural diversity. In summary, the measurement of functional responses using voltage-sensitive dyes was flexible and cost-effective because labelled competitors were not needed, effects were independent of a specific binding site, and both agonist and antagonist actions were probed in a single assay. The method is widely applicable and could be applied to all P2X family members, as well as other voltage-gated and ligand-gated ion channels. This article is part of the Special Issue entitled ‘Fluorescent Tools in Neuropharmacology’.
A novel fluorescence-based screening approach for identifying P2X1 receptor ligand candidates. Fragment-based drug discovery applied to ligand-gated ion channels. The use of confocal microscopy to determine the kinetics and affinity of Alexa-647-ATP binding to P2X1 receptors. Alexa-647-ATP for imaging P2X1 receptors on live cells.
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Affiliation(s)
- Marc-David Ruepp
- Department of Chemistry and Biochemistry, University of Bern, Freiestrasse 3, CH-3012 Bern, Switzerland
| | - James A Brozik
- Washington State University, Department of Chemistry, Pullman, WA 99164-4630, USA
| | - Iwan J P de Esch
- Medicinal Chemistry, VU University Amsterdam, Amsterdam, The Netherlands
| | - Richard W Farndale
- Department of Biochemistry, University of Cambridge, Cambridge CB2 1QW, UK
| | | | - Andrew J Thompson
- Department of Pharmacology, Tennis Court Road, Cambridge CB2 1PD, UK.
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20
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Sophocleous RA, Mullany PRF, Winter KM, Marks DC, Sluyter R. Propensity of red blood cells to undergo P2X7 receptor-mediated phosphatidylserine exposure does not alter during in vivo or ex vivo aging. Transfusion 2015; 55:1946-54. [PMID: 25823581 DOI: 10.1111/trf.13101] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/12/2014] [Revised: 02/13/2015] [Accepted: 02/13/2015] [Indexed: 12/20/2022]
Abstract
BACKGROUND Phosphatidylserine (PS) exposure facilitates the removal of red blood cells (RBCs) from the circulation, potentially contributing to the loss of stored RBCs after transfusion, as well as senescent RBCs. Activation of the P2X7 receptor by extracellular adenosine 5'-triphosphate (ATP) can induce PS exposure on freshly isolated human RBCs, but whether this process occurs in stored RBCs or changes during RBC aging is unknown. STUDY DESIGN AND METHODS RBCs were processed and stored according to Australian blood banking guidelines. PS exposure was determined by annexin V binding and flow cytometry. Efficacy of P2X antagonists was assessed by flow cytometric measurements of ATP-induced ethidium+ uptake in RPMI 8226 cells. Osmotic fragility was assessed by lysis in hypotonic saline. RBCs were fractionated by discontinuous density centrifugation. RESULTS ATP (1 mmol/L) induced PS exposure on RBCs stored for less than 1 week. This process was near-completely inhibited by the P2X7 antagonists A438079 and AZ10606120 and the P2X1/P2X7 antagonist MRS2159 but not the P2X1 antagonist NF499. ATP-induced PS exposure on RBCs was not dependent on K+, Na+, or Cl- fluxes. ATP did not alter the osmotic fragility of stored RBCs. ATP-induced PS exposure was similar between RBCs of different densities. ATP-induced PS exposure was also similar between RBCs stored for less than 1 week or for 6 weeks. CONCLUSION The propensity of RBCs to undergo P2X7-mediated PS exposure does not alter during in vivo and ex vivo aging. Thus, P2X7 activation is unlikely to be involved in the removal of senescent RBCs or stored RBCs after transfusion.
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Affiliation(s)
- Reece A Sophocleous
- School of Biological Sciences.,Centre for Medical and Molecular Bioscience, University of Wollongong.,Illawarra Health and Medical Research Institute, Wollongong, NSW, Australia
| | - Phillip R F Mullany
- School of Biological Sciences.,Centre for Medical and Molecular Bioscience, University of Wollongong.,Illawarra Health and Medical Research Institute, Wollongong, NSW, Australia
| | - Kelly M Winter
- Research and Development, Australian Red Cross Blood Service, Sydney, NSW, Australia
| | - Denese C Marks
- Research and Development, Australian Red Cross Blood Service, Sydney, NSW, Australia
| | - Ronald Sluyter
- School of Biological Sciences.,Centre for Medical and Molecular Bioscience, University of Wollongong.,Illawarra Health and Medical Research Institute, Wollongong, NSW, Australia
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Michel MC, Seifert R. Selectivity of pharmacological tools: implications for use in cell physiology. A review in the theme: Cell signaling: proteins, pathways and mechanisms. Am J Physiol Cell Physiol 2015; 308:C505-20. [PMID: 25631871 DOI: 10.1152/ajpcell.00389.2014] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/08/2014] [Accepted: 01/24/2015] [Indexed: 01/08/2023]
Abstract
Pharmacological inhibitors are frequently used to identify the receptors, receptor subtypes, and associated signaling pathways involved in physiological cell responses. Based on the effects of such inhibitors conclusions are drawn about the involvement of their assumed target or lack thereof. While such inhibitors can be useful tools for a better physiological understanding, their uncritical use can lead to incorrect conclusions. This article reviews the concept of inhibitor selectivity and its implication for cell physiology. Specifically, we discuss the implications of using inhibitor vs. activator approaches, issues of direct vs. indirect pathway modulation, implications of inverse agonism and biased signaling, and those of orthosteric vs. allosteric, competitive vs. noncompetitive, and reversible vs. irreversible inhibition. Additional problems can result from inconsistent estimates of inhibitor potency and differences in potency between cell-free systems and intact cells. These concepts are illustrated by several examples of inhibitors displaying affinity for related but distinct targets or even unrelated targets. Of note, many of the issues being addressed are also applicable to genetic inhibition strategies. The main practical conclusion following from these concepts is that investigators should be critical in the choice of inhibitor, its concentrations, and its mode of application. When this advice is adhered to, small-molecule pharmacological inhibitors can be important experimental tools in the hand of physiologists.
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Affiliation(s)
- Martin C Michel
- Department of Pharmacology, Johannes Gutenberg University, Mainz, Germany; and
| | - Roland Seifert
- Department of Pharmacology, Hannover Medical School, Hannover, Germany
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Farmer LK, Schmid R, Evans RJ. Use of chimeras, point mutants, and molecular modeling to map the antagonist-binding site of 4,4',4″,4‴-(carbonylbis-(imino-5,1,3-benzenetriylbis(carbonylimino)))tetrakisbenzene-1,3-disulfonic acid (NF449) at P2X1 receptors for ATP. J Biol Chem 2014; 290:1559-69. [PMID: 25425641 PMCID: PMC4340402 DOI: 10.1074/jbc.m114.592246] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/16/2023] Open
Abstract
P2X receptor subtype-selective antagonists are promising candidates for treatment of a range of pathophysiological conditions. However, in contrast to high resolution structural understanding of agonist action in the receptors, comparatively little is known about the molecular basis of antagonist binding. We have generated chimeras and point mutations in the extracellular ligand-binding loop of the human P2X1 receptor, which is inhibited by NF449, suramin, and pyridoxal-phosphate-6-azophenyl-2,4-disulfonate, with residues from the rat P2X4 receptor, which is insensitive to these antagonists. There was little or no effect on sensitivity to suramin and pyridoxal-phosphate-6-azophenyl-2,4-disulfonate in chimeric P2X1/4 receptors, indicating that a significant number of residues required for binding of these antagonists are present in the P2X4 receptor. Sensitivity to the P2X1 receptor-selective antagonist NF449 was reduced by ∼60- and ∼135-fold in chimeras replacing the cysteine-rich head, and the dorsal fin region below it in the adjacent subunit, respectively. Point mutants identified the importance of four positively charged residues at the base of the cysteine-rich head and two variant residues in the dorsal fin for high affinity NF449 binding. These six residues were used as the starting area for molecular docking. The four best potential NF449-binding poses were then discriminated by correspondence with the mutagenesis data and an additional mutant to validate the binding of one lobe of NF449 within the core conserved ATP-binding pocket and the other lobes coordinated by positive charge on the cysteine-rich head region and residues in the adjacent dorsal fin.
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Affiliation(s)
- Louise K Farmer
- From the Departments of Cell Physiology and Pharmacology and
| | - Ralf Schmid
- Biochemistry, University of Leicester, Leicester LE1 9HN, United Kingdom
| | - Richard J Evans
- From the Departments of Cell Physiology and Pharmacology and
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Purinergic P2X receptors: structural models and analysis of ligand-target interaction. Eur J Med Chem 2014; 89:561-80. [PMID: 25462266 DOI: 10.1016/j.ejmech.2014.10.071] [Citation(s) in RCA: 36] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/01/2014] [Revised: 10/07/2014] [Accepted: 10/24/2014] [Indexed: 01/04/2023]
Abstract
The purinergic P2X receptors are ligand-gated cation channels activated by the endogenous ligand ATP. They assemble as homo- or heterotrimers from seven cloned subtypes (P2X1-7) and all trimer subunits present a common topology consisting in intracellular N- and C- termini, two transmembrane domains and a large extracellular domain. These membrane proteins are present in virtually all mammalian tissues and regulate a large variety of responses in physio- and pathological conditions. The development of ligands that selectively activate or block specific P2X receptor subtypes hence represents a promising strategy to obtain novel pharmacological tools for the treatment of pain, cancer, inflammation, and neurological, cardiovascular, and endocrine diseases. The publication of the crystal structures of zebrafish P2X4 receptor in inactive and ATP-bound active forms provided structural data for the analysis of the receptor structure, the interpretation of mutagenesis data, and the depiction of ligand binding and receptor activation mechanism. In addition, the availability of ATP-competitive ligands presenting selectivity for P2X receptor subtypes supports the design of new potent and selective ligands with possibly improved pharmacokinetic profiles, with the final aim to obtain new drugs. This study describes molecular modelling studies performed to develop structural models of the human and rat P2X receptors in inactive and active states. These models allowed to analyse the role of some non-conserved residues at ATP binding site and to study the receptor interaction with some non-specific or subtype selective agonists and antagonists.
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24
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Horakova D, Cela P, Krejci P, Balek L, Moravcova Balkova S, Matalova E, Buchtova M. Effect of FGFR inhibitors on chicken limb development. Dev Growth Differ 2014; 56:555-72. [DOI: 10.1111/dgd.12156] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/01/2013] [Revised: 07/30/2014] [Accepted: 08/07/2014] [Indexed: 11/26/2022]
Affiliation(s)
- Dana Horakova
- Department of Anatomy, Histology and Embryology; Faculty of Veterinary Medicine; University of Veterinary and Pharmaceutical Sciences; Brno Czech Republic
| | - Petra Cela
- Institute of Animal Physiology and Genetics, v.v.i.; Academy of Sciences of the Czech Republic; Brno Czech Republic
- Department of Animal Physiology and Immunology; Institute of Experimental Biology; Masaryk University; Brno Czech Republic
| | - Pavel Krejci
- Department of Animal Physiology and Immunology; Institute of Experimental Biology; Masaryk University; Brno Czech Republic
- Department of Biology; Faculty of Medicine; Masaryk University; Brno Czech Republic
| | - Lukas Balek
- Department of Animal Physiology and Immunology; Institute of Experimental Biology; Masaryk University; Brno Czech Republic
- Department of Biology; Faculty of Medicine; Masaryk University; Brno Czech Republic
| | - Simona Moravcova Balkova
- Institute of Animal Physiology and Genetics, v.v.i.; Academy of Sciences of the Czech Republic; Brno Czech Republic
- Clinic of Stomatology, St. Anne's Faculty Hospital and Faculty of Medicine; Masaryk University; Brno Czech Republic
| | - Eva Matalova
- Institute of Animal Physiology and Genetics, v.v.i.; Academy of Sciences of the Czech Republic; Brno Czech Republic
- Department of Physiology; Faculty of Veterinary Medicine; University of Veterinary and Pharmaceutical Sciences; Brno Czech Republic
| | - Marcela Buchtova
- Department of Anatomy, Histology and Embryology; Faculty of Veterinary Medicine; University of Veterinary and Pharmaceutical Sciences; Brno Czech Republic
- Institute of Animal Physiology and Genetics, v.v.i.; Academy of Sciences of the Czech Republic; Brno Czech Republic
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Vascular smooth muscle cells from small human omental arteries express P2X1 and P2X4 receptor subunits. Purinergic Signal 2014; 10:565-72. [PMID: 24845338 DOI: 10.1007/s11302-014-9415-6] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/24/2014] [Accepted: 05/08/2014] [Indexed: 02/06/2023] Open
Abstract
Stimulation of P2X receptors by ATP in vascular smooth muscle cells (VSMCs) is proposed to mediate vascular tone. However, understanding of P2X receptor-mediated actions in human blood vessels is limited, and therefore, the current work investigates the role of P2X receptors in freshly isolated small human gastro-omental arteries (HGOAs). Expression of P2X1 and P2X4 receptor subunit messenger RNA (mRNA) and protein was identified in individual HGOA VSMCs using RT-PCR and immunofluorescent analysis and using Western blot in multi-cellular preparations. ATP of 10 μmol/l and αβ-meATP of 10 μmol/l, a selective P2X receptor agonist, evoked robust increases in [Ca(2+)]i in fluo-3-loaded HGOA VSMCs. Pre-incubation with 1 μmol/l NF279, a selective P2X receptor antagonist, reduced the amplitude of αβ-meATP-induced increase in [Ca(2+)]i by about 70 %. ATP of 10 μmol/l and αβ-meATP of 10 μmol/l produced similar contractile responses in segments of HGOA, and these contractions were greatly reduced by 2 μmol/l NF449, a selective P2X receptor inhibitor. These data suggest that VSMCs from HGOA express P2X1 and P2X4 receptor subunits with homomeric P2X1 receptors likely serving as the predominant target for extracellular ATP.
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Kaczmarek-Hájek K, Lörinczi E, Hausmann R, Nicke A. Molecular and functional properties of P2X receptors--recent progress and persisting challenges. Purinergic Signal 2012; 8:375-417. [PMID: 22547202 PMCID: PMC3360091 DOI: 10.1007/s11302-012-9314-7] [Citation(s) in RCA: 176] [Impact Index Per Article: 13.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/02/2011] [Accepted: 10/18/2011] [Indexed: 12/16/2022] Open
Abstract
ATP-gated P2X receptors are trimeric ion channels that assemble as homo- or heteromers from seven cloned subunits. Transcripts and/or proteins of P2X subunits have been found in most, if not all, mammalian tissues and are being discovered in an increasing number of non-vertebrates. Both the first crystal structure of a P2X receptor and the generation of knockout (KO) mice for five of the seven cloned subtypes greatly advanced our understanding of their molecular and physiological function and their validation as drug targets. This review summarizes the current understanding of the structure and function of P2X receptors and gives an update on recent developments in the search for P2X subtype-selective ligands. It also provides an overview about the current knowledge of the regulation and modulation of P2X receptors on the cellular level and finally on their physiological roles as inferred from studies on KO mice.
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Affiliation(s)
- Karina Kaczmarek-Hájek
- Max Planck Institute for Experimental Medicine, Hermann Rein Str. 3, 37075, Göttingen, Germany
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El-Ajouz S, Ray D, Allsopp RC, Evans RJ. Molecular basis of selective antagonism of the P2X1 receptor for ATP by NF449 and suramin: contribution of basic amino acids in the cysteine-rich loop. Br J Pharmacol 2012; 165:390-400. [PMID: 21671897 PMCID: PMC3268193 DOI: 10.1111/j.1476-5381.2011.01534.x] [Citation(s) in RCA: 37] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022] Open
Abstract
BACKGROUND AND PURPOSE The cysteine-rich head region, which is adjacent to the proposed ATP-binding pocket in the extracellular ligand-binding loop of P2X receptors for ATP, is absent in the antagonist-insensitive Dictyostelium receptors. In this study we have determined the contribution of the head region to the antagonist action of NF449 and suramin at the human P2X1 receptor. EXPERIMENTAL APPROACH Chimeras and point mutations in the cysteine-rich head region were made between human P2X1 and P2X2 receptors. Mutant receptors were expressed in Xenopus oocytes and P2X receptor currents characterized using two-electrode voltage clamp. KEY RESULTS The chimera replacing the region between the third and fourth conserved cysteine residues of the P2X1 receptor with the corresponding part of P2X2 reduced NF449 sensitivity a thousand fold from an IC50 of ∼1 nM at the P2X1 receptor to that of the P2X2 receptor (IC50∼1 µM). A similar decrease in sensitivity resulted from mutation of four positively charged P2X1 receptor residues in this region that are absent from the P2X2 receptor. These chimeras and mutations were also involved in determining sensitivity to the antagonist suramin. Reciprocal chimeras and mutations in the P2X2 receptor produced modest increases in antagonist sensitivity. CONCLUSIONS AND IMPLICATIONS These data indicate that a cluster of positively charged residues at the base of the cysteine-rich head region can account for the highly selective antagonism of the P2X1 receptor by the suramin derivative NF449.
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Affiliation(s)
- S El-Ajouz
- Department of Cell Physiology and Pharmacology, University of Leicester, Leicester, UK
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28
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Shirakawa T, Nakano K, Hachiya N, Kato N, Kaneko K. The involvement of P2X1 receptor in pyramidal cell degeneration in the rat hippocampus after trimethyltin administration. Neurosci Res 2011; 71:396-404. [DOI: 10.1016/j.neures.2011.08.005] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/22/2011] [Revised: 07/20/2011] [Accepted: 08/10/2011] [Indexed: 12/14/2022]
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Coddou C, Yan Z, Obsil T, Huidobro-Toro JP, Stojilkovic SS. Activation and regulation of purinergic P2X receptor channels. Pharmacol Rev 2011; 63:641-83. [PMID: 21737531 PMCID: PMC3141880 DOI: 10.1124/pr.110.003129] [Citation(s) in RCA: 405] [Impact Index Per Article: 28.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022] Open
Abstract
Mammalian ATP-gated nonselective cation channels (P2XRs) can be composed of seven possible subunits, denoted P2X1 to P2X7. Each subunit contains a large ectodomain, two transmembrane domains, and intracellular N and C termini. Functional P2XRs are organized as homomeric and heteromeric trimers. This review focuses on the binding sites involved in the activation (orthosteric) and regulation (allosteric) of P2XRs. The ectodomains contain three ATP binding sites, presumably located between neighboring subunits and formed by highly conserved residues. The detection and coordination of three ATP phosphate residues by positively charged amino acids are likely to play a dominant role in determining agonist potency, whereas an AsnPheArg motif may contribute to binding by coordinating the adenine ring. Nonconserved ectodomain histidines provide the binding sites for trace metals, divalent cations, and protons. The transmembrane domains account not only for the formation of the channel pore but also for the binding of ivermectin (a specific P2X4R allosteric regulator) and alcohols. The N- and C- domains provide the structures that determine the kinetics of receptor desensitization and/or pore dilation and are critical for the regulation of receptor functions by intracellular messengers, kinases, reactive oxygen species and mercury. The recent publication of the crystal structure of the zebrafish P2X4.1R in a closed state provides a major advance in the understanding of this family of receptor channels. We will discuss data obtained from numerous site-directed mutagenesis experiments accumulated during the last 15 years with reference to the crystal structure, allowing a structural interpretation of the molecular basis of orthosteric and allosteric ligand actions.
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Affiliation(s)
- Claudio Coddou
- Section on Cellular Signaling, Program in Developmental Neuroscience, National Institute of Child Health and Human Developmant, National Institutes of Health, Bethesda, MD 20892-4510, USA
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30
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Wolf C, Rosefort C, Fallah G, Kassack MU, Hamacher A, Bodnar M, Wang H, Illes P, Kless A, Bahrenberg G, Schmalzing G, Hausmann R. Molecular determinants of potent P2X2 antagonism identified by functional analysis, mutagenesis, and homology docking. Mol Pharmacol 2011; 79:649-61. [PMID: 21191044 DOI: 10.1124/mol.110.068700] [Citation(s) in RCA: 38] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/25/2023] Open
Abstract
P2X2 receptors are members of the ATP-gated P2X family of cation channels, and they participate in neurotransmission in sympathetic ganglia and interneurons. Here, we identified 7,7'-(carbonylbis(imino-3,1-phenylenecarbonylimino-3,1-(4-methyl-phenylene)carbonylimino))bis(1-methoxy-naphthalene-3,6-disulfonic acid) tetrasodium salt (NF770) as a nanomolar-potent competitive P2X2 receptor antagonist within a series of 139 suramin derivatives. Three structural determinants contributed to the inhibition of P2X2 receptors by NF770: 1) a "large urea" structure with two symmetric phenylenecarbonylimino groups; 2) attachment of the naphthalene moiety in position 7,7'; and 3) the specific position of two sulfonic acid groups (3,3'; 6,6') and of one methoxy group (1,1') at the naphthalene moiety. This structure-activity relationship was interpreted using a rat P2X2 homology model based on the crystal structure of the closed zebrafish P2X4 receptor. Docking of the suramin derivatives into the modeled ATP-binding pocket provides a uniform explanation for the observed differences in inhibitory potencies. Changes in the chemical structure that increase the inhibitory potency of the suramin derivatives improved the spatial orientation within the ATP-binding pocket to allow for stronger polar interactions of functional groups with Gly72, Glu167, or Arg290. Gly72 is responsible for the orientation of the methoxy group close to Arg290 or Glu167. Combined mutational and functional analysis confirmed that residues Gly72 and Glu167 are as important for ATP binding as Arg290, the ATP-binding role of which has been shown in previous studies. The in silico prediction of Gly72 and Glu167 as ATP-binding residues strongly supports the validity of our homology docking.
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Affiliation(s)
- Christian Wolf
- Department of Molecular Pharmacology, RWTH Aachen University, Aachen, Germany
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31
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Su TR, Hung YS, Huang SS, Su HH, Su CC, Hsiao G, Chen YH, Lin MJ. Study of the reversal effect of NF449 on neuromuscular blockade induced by d-tubocurarine. Life Sci 2011; 88:1039-46. [PMID: 21453711 DOI: 10.1016/j.lfs.2011.03.013] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/05/2010] [Revised: 02/23/2011] [Accepted: 03/22/2011] [Indexed: 12/01/2022]
Abstract
AIMS The aim of this study was to investigate the mechanism for the reversal effect of NF449 (a suramin analogue) on the neuromuscular block induced by d-tubocurarine (d-TC). MAIN METHODS Nerve-stimulated muscle contractions and end-plate potentials were performed in mouse phrenic nerve-diaphragm preparations. Acetylcholine (ACh)-induced muscle contractions were performed in the chick biventer cervicis preparations. Presynaptic nerve terminal waveform recordings were performed in mouse triangularis sterni preparations. KEY FINDINGS Amongst the suramin analogues in this study, only the NF449 and suramin were able to reverse the blockade effect produced by d-TC on nerve-stimulated muscle contractions. Each of these suramin analogues (NF007, NF023, NF279 and NF449) alone has no significant effect on the amplitude of nerve-stimulated muscle contractions. NF449 and suramin also showed the antagonising effects on the inhibition of end-plate potentials induced by d-TC. Furthermore, pre-treatment with NF449 can antagonise the inhibition of d-TC in ACh-induced contractions of chick biventer cervicis muscle. NF449 produced a greater rightward shift of the dose-response inhibition curve for d-TC than did suramin. Because other purinergic 2X (P2X) receptor antagonists, NF023 and NF279, do not have the reverse effects on the neuromuscular blockade of d-TC, the effect of NF449 seems irrelevant to inhibition of P2X receptors. SIGNIFICANCE These data suggest that NF449 was able to compete with the binding of d-TC on the nicotinic ACh receptors, and the effect of NF449 was more potent than suramin in reducing the inhibition of d-TC. The structure of NF449 may provide useful information for designing potent antidotes against neuromuscular toxins.
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Affiliation(s)
- Tzu-Rong Su
- Tian-Sheng Memorial Hospital, Tong-Kang, Pintong, Taiwan
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32
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Mahaut-Smith MP, Jones S, Evans RJ. The P2X1 receptor and platelet function. Purinergic Signal 2011; 7:341-56. [PMID: 21484087 PMCID: PMC3166991 DOI: 10.1007/s11302-011-9224-0] [Citation(s) in RCA: 45] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/23/2010] [Accepted: 02/22/2011] [Indexed: 12/17/2022] Open
Abstract
Extracellular nucleotides are ubiquitous signalling molecules, acting via the P2 class of surface receptors. Platelets express three P2 receptor subtypes, ADP-dependent P2Y1 and P2Y12 G-protein-coupled receptors and the ATP-gated P2X1 non-selective cation channel. Platelet P2X1 receptors can generate significant increases in intracellular Ca(2+), leading to shape change, movement of secretory granules and low levels of α(IIb)β(3) integrin activation. P2X1 can also synergise with several other receptors to amplify signalling and functional events in the platelet. In particular, activation of P2X1 receptors by ATP released from dense granules amplifies the aggregation responses to low levels of the major agonists, collagen and thrombin. In vivo studies using transgenic murine models show that P2X1 receptors amplify localised thrombosis following damage of small arteries and arterioles and also contribute to thromboembolism induced by intravenous co-injection of collagen and adrenaline. In vitro, under flow conditions, P2X1 receptors contribute more to aggregate formation on collagen-coated surfaces as the shear rate is increased, which may explain their greater contribution to localised thrombosis in arterioles compared to venules within in vivo models. Since shear increases substantially near sites of stenosis, anti-P2X1 therapy represents a potential means of reducing thrombotic events at atherosclerotic plaques.
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Affiliation(s)
- Martyn P Mahaut-Smith
- Department of Cell Physiology and Pharmacology, University of Leicester, Leicester, LE1 9HN, UK,
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Abstract
P2X receptors for ATP are ligand gated cation channels that form from the trimeric assembly of subunits with two transmembrane segments, a large extracellular ligand binding loop, and intracellular amino and carboxy termini. The receptors are expressed throughout the body, involved in functions ranging from blood clotting to inflammation, and may provide important targets for novel therapeutics. Mutagenesis based studies have been used to develop an understanding of the molecular basis of their pharmacology with the aim of developing models of the ligand binding site. A crystal structure for the zebra fish P2X4 receptor in the closed agonist unbound state has been published recently, which provides a major advance in our understanding of the receptors. This review gives an overview of mutagenesis studies that have led to the development of a model of the ATP binding site, as well as identifying residues contributing to allosteric regulation and antagonism. These studies are discussed with reference to the crystal to provide a structural interpretation of the molecular basis of drug action.
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Affiliation(s)
- Richard J Evans
- Cell Physiology & Pharmacology, University of Leicester, Leicester, UK.
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Baqi Y, Hausmann R, Rosefort C, Rettinger J, Schmalzing G, Müller CE. Discovery of potent competitive antagonists and positive modulators of the P2X2 receptor. J Med Chem 2011; 54:817-30. [PMID: 21207957 DOI: 10.1021/jm1012193] [Citation(s) in RCA: 52] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/03/2023]
Abstract
Evaluation and optimization of anthraquinone derivatives related to Reactive Blue 2 at P2X2 receptors yielded the first potent and selective P2X2 receptor antagonists. The compounds were tested for inhibition of ATP (10 μM) mediated currents in Xenopus oocytes expressing the rat P2X2 receptor. The most potent antagonists were sodium 1-amino-4-[3-(4,6-dichloro[1,3,5]triazine-2-ylamino)phenylamino]-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate (63, PSB-10211, IC(50) 86 nM) and disodium 1-amino-4-[3-(4,6-dichloro[1,3,5]triazine-2-ylamino)-4-sulfophenylamino]-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate (57, PSB-1011, IC(50) 79 nM). Compound 57 exhibited a competitive mechanism of action (pA(2) 7.49). It was >100-fold selective versus P2X4, P2X7, and several investigated P2Y receptor subtypes (P2Y(2,4,6,12)); selectivity versus P2X1 and P2X3 receptors was moderate (>5-fold). Compound 57 was >13-fold more potent at the homomeric P2X2 than at the heteromeric P2X2/3 receptor. Several anthraquinone derivatives were found to act as positive modulators of ATP effects at P2X2 receptors, for example, sodium 1-amino-4-(3-phenoxyphenylamino)-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate (51, PSB-10129, EC(50) 489 nM), which led to about a 3-fold increase in the ATP-elicited current.
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Affiliation(s)
- Younis Baqi
- PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, Pharmaceutical Sciences Bonn (PSB), University of Bonn, An der Immenburg 4, D-53121 Bonn, Germany
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35
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Characterisation of P2X receptors expressed in rat pulmonary arteries. Eur J Pharmacol 2010; 649:342-8. [DOI: 10.1016/j.ejphar.2010.09.041] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/28/2010] [Revised: 08/27/2010] [Accepted: 09/07/2010] [Indexed: 12/31/2022]
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Krejci P, Murakami S, Prochazkova J, Trantirek L, Chlebova K, Ouyang Z, Aklian A, Smutny J, Bryja V, Kozubik A, Wilcox WR. NF449 is a novel inhibitor of fibroblast growth factor receptor 3 (FGFR3) signaling active in chondrocytes and multiple myeloma cells. J Biol Chem 2010; 285:20644-53. [PMID: 20439987 PMCID: PMC2898326 DOI: 10.1074/jbc.m109.083626] [Citation(s) in RCA: 33] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/10/2009] [Revised: 03/31/2010] [Indexed: 11/06/2022] Open
Abstract
The FGFR3 receptor tyrosine kinase represents an attractive target for therapy due to its role in several human disorders, including skeletal dysplasias, multiple myeloma, and cervical and bladder carcinomas. By using molecular library screening, we identified a compound named NF449 with inhibitory activity toward FGFR3 signaling. In cultured chondrocytes and murine limb organ culture, NF449 rescued FGFR3-mediated extracellular matrix loss and growth inhibition, which represent two major cellular phenotypes of aberrant FGFR3 signaling in cartilage. Similarly, NF449 antagonized FGFR3 action in the multiple myeloma cell lines OPM2 and KMS11, as evidenced by NF449-mediated reversal of ERK MAPK activation and transcript accumulation of CCL3 and CCL4 chemokines, both of which are induced by FGFR3 activation. In cell-free kinase assays, NF449 inhibited the kinase activity of both wild type and a disease-associated FGFR3 mutant (K650E) in a fashion that appeared non-competitive with ATP. Our data identify NF449 as a novel antagonist of FGFR3 signaling, useful for FGFR3 inhibition alone or in combination with inhibitors that target the ATP binding site.
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Affiliation(s)
- Pavel Krejci
- Medical Genetics Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA.
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37
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Meis S, Hamacher A, Hongwiset D, Marzian C, Wiese M, Eckstein N, Royer HD, Communi D, Boeynaems JM, Hausmann R, Schmalzing G, Kassack MU. NF546 [4,4'-(carbonylbis(imino-3,1-phenylene-carbonylimino-3,1-(4-methyl-phenylene)-carbonylimino))-bis(1,3-xylene-alpha,alpha'-diphosphonic acid) tetrasodium salt] is a non-nucleotide P2Y11 agonist and stimulates release of interleukin-8 from human monocyte-derived dendritic cells. J Pharmacol Exp Ther 2010; 332:238-47. [PMID: 19815812 DOI: 10.1124/jpet.109.157750] [Citation(s) in RCA: 82] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023] Open
Abstract
The G protein-coupled P2Y(11) receptor is involved in immune system modulation. In-depth physiological evaluation is hampered, however, by a lack of selective and potent ligands. By screening a library of sulfonic and phosphonic acid derivatives at P2Y(11) receptors recombinantly expressed in human 1321N1 astrocytoma cells (calcium and cAMP assays), the selective non-nucleotide P2Y(11) agonist NF546 [4,4'-(carbonylbis(imino-3,1-phenylene-carbonylimino-3,1-(4-methyl-phenylene)carbonylimino))-bis(1,3-xylene-alpha,alpha'-diphosphonic acid) tetrasodium salt] was identified. NF546 had a pEC(50) of 6.27 and is relatively selective for P2Y(11) over P2Y(1), P2Y(2), P2Y(4), P2Y(6), P2Y(12), P2X(1), P2X(2), and P2X(2)-X(3). Adenosine-5'-O-(3-thio)triphosphate (ATPgammaS), a nonhydrolyzable analog of the physiological P2Y(11) agonist ATP, and NF546 use a common binding site as suggested by molecular modeling studies and their competitive behavior toward the nanomolar potency antagonist NF340 [4,4'-(carbonylbis(imino-3,1-(4-methyl-phenylene)carbonylimino))bis(naphthalene-2,6-disulfonic acid) tetrasodium salt] in Schild analysis. The pA(2) of NF340 was 8.02 against ATPgammaS and 8.04 against NF546 (calcium assays). NF546 was further tested for P2Y(11)-mediated effects in monocyte-derived dendritic cells. Similarly to ATPgammaS, NF546 led to thrombospondin-1 secretion and inhibition of lipopolysaccharide-stimulated interleukin-12 release, whereas NF340 inhibited these effects. Further, for the first time, it was shown that ATPgammaS or NF546 stimulation promotes interleukin 8 (IL-8) release from dendritic cells, which could be inhibited by NF340. In conclusion, we have described the first selective, non-nucleotide agonist NF546 for P2Y(11) receptors in both recombinant and physiological expression systems and could show a P2Y(11)-stimulated IL-8 release, further supporting the immunomodulatory role of P2Y(11) receptors.
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Affiliation(s)
- Sabine Meis
- Institute of Pharmaceutical and Medicinal Chemistry, Pharmaceutical Biochemistry, Heinrich-Heine-University of Duesseldorf, Duesseldorf, Germany
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Sim JA, Broomhead HE, North RA. Ectodomain lysines and suramin block of P2X1 receptors. J Biol Chem 2008; 283:29841-6. [PMID: 18765669 PMCID: PMC2573084 DOI: 10.1074/jbc.m802523200] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/01/2008] [Revised: 08/11/2008] [Indexed: 11/22/2022] Open
Abstract
P2X(1) receptors belong to a family of cation channels gated by extracellular ATP; they are found inter alia in smooth muscle, platelets, and immune cells. Suramin has been widely used as an antagonist at P2X receptors, and its analog 4,4',4'',4'''-[carbonylbis(imino-5,1,3-benzenetriylbis(carbonylimino))] tetrakis-benzene-1,3-disulfonic acid (NF449) is selective for the P2X(1) subtype. Human and mouse P2X(1) receptors were expressed in human embryonic kidney cells, and membrane currents evoked by ATP were recorded. ATP (10 nm to 100 microm) was applied only once to each cell, to avoid the profound desensitization exhibited by P2X(1) receptors. Suramin (10 microm) and NF449 (3-300 nM) effectively blocked the human receptor. Suramin had little effect on the mouse receptor. Suramin and NF449 are polysulfonates, with six and eight negative charges, respectively. We hypothesized that species differences might result from differences in positive residues presented by the large receptor ectodomain. Four lysines in the human sequence (Lys(111), Lys(127), Lys(138), and Lys(148)) were changed individually and together to their counterparts in the mouse sequence. The substitution K138E, either alone or together with K111Q, K127Q, and K148N, reduced the sensitivity to block by both suramin and NF449. Conversely, when lysine was introduced into the mouse receptor, the sensitivity to block by suramin and NF449 was much increased for E138K, but not for Q111K, Q127K, or N148K. The results explain the marked species difference in antagonist sensitivity and identify an ectodomain lysine residue that plays a key role in the binding of both suramin and NF449 to P2X(1) receptors.
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Affiliation(s)
- Joan A Sim
- Faculty of Life Sciences, University of Manchester, Michael Smith Building, Oxford Road, Manchester M13 9PT, United Kingdom.
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Targeted disruption of the A2A adenosine receptor reduces in-vitro prostate contractility in mature mice. Eur J Pharmacol 2008; 592:151-7. [DOI: 10.1016/j.ejphar.2008.07.003] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/02/2008] [Revised: 06/04/2008] [Accepted: 07/02/2008] [Indexed: 11/20/2022]
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Heinrich A, Kittel A, Csölle C, Sylvester Vizi E, Sperlágh B. Modulation of neurotransmitter release by P2X and P2Y receptors in the rat spinal cord. Neuropharmacology 2007; 54:375-86. [PMID: 18063000 DOI: 10.1016/j.neuropharm.2007.10.013] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/16/2007] [Revised: 09/28/2007] [Accepted: 10/15/2007] [Indexed: 12/22/2022]
Abstract
In this study, the P2 receptor-mediated modulation of [3H]glutamate and [3H]noradrenaline release were examined in rat spinal cord slices. Adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP), and 2-methylthioadenosine 5'-diphosphate (2-MeSADP) decreased the electrical stimulation-evoked [3H]glutamate efflux with the following order of potency: ADP>2-MeSADP>ATP. The effect of ATP was antagonized by suramin (300microM), the P2Y12,13 receptor antagonist 2-methylthioadenosine 5'-monophosphate (2-MeSAMP, 10microM), and partly by 4-[[4-Formyl-5-hydroxy-6-methyl-3-[(phosphonooxy)methyl]-2-pyridinyl]azo]-1,3-benzenedisulfonic acid (PPADS, 30microM) and the P2Y1 receptor antagonist 2'-deoxy-N6-methyladenosine 3',5'-diphosphate (MRS 2179, 10muM). ATP, ADP and 2-MeSADP also decreased evoked [3H]noradrenaline outflow; the order of agonist potency was ADP> or =2-MeSADP>ATP. The effect of ATP was reversed by 2-MeSAMP (10microM), and partly by MRS 2179 (10microM). By contrast, 2-methylthioadenosine-5'-triphosphate (2-MeSATP, 10-300microM) increased resting and electrically evoked [3H]glutamate and [3H]noradrenaline efflux, and this effect was prevented by the P2X1 receptor selective antagonist 4,4',4'',4'''-[carbonylbis[imino-5,1,3-benzenetriyl bis (carbonyl-imino)]] tetrakis (benzene-1,3-disulfonic acid) octasodium salt (NF449, 100nM). Reverse transcriptase polymerase chain reaction (RT-PCR) analysis revealed that mRNAs encoding P2Y12 and P2Y13 receptors are expressed in the brainstem, whereas P2Y13 but not P2Y12 receptor mRNA is present in the dorsal root ganglion and spinal cord. P2Y1 receptor expression in the spinal cord is also demonstrated at the protein level. In conclusion, inhibitory P2Y and facilitatory P2X1-like receptors, involved in the regulation of glutamate (P2Y13 and/or P2Y1) and noradrenaline (P2Y13 and/or P2Y1, P2Y12) release have been identified, which provide novel target sites for analgesics acting at the spinal cord level.
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Affiliation(s)
- Attila Heinrich
- Laboratory of Molecular Pharmacology, Institute of Experimental Medicine, Hungarian Academy of Sciences, H-1450 Budapest POB 67, Hungary
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Munkonda MN, Kauffenstein G, Kukulski F, Lévesque SA, Legendre C, Pelletier J, Lavoie EG, Lecka J, Sévigny J. Inhibition of human and mouse plasma membrane bound NTPDases by P2 receptor antagonists. Biochem Pharmacol 2007; 74:1524-34. [PMID: 17727821 DOI: 10.1016/j.bcp.2007.07.033] [Citation(s) in RCA: 45] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/30/2007] [Revised: 07/21/2007] [Accepted: 07/23/2007] [Indexed: 11/15/2022]
Abstract
The plasma membrane bound nucleoside triphosphate diphosphohydrolase (NTPDase)-1, 2, 3 and 8 are major ectonucleotidases that modulate P2 receptor signaling by controlling nucleotides' concentrations at the cell surface. In this report, we systematically evaluated the effect of the commonly used P2 receptor antagonists reactive blue 2, suramin, NF279, NF449 and MRS2179, on recombinant human and mouse NTPDase1, 2, 3 and 8. Enzymatic reactions were performed in a Tris/calcium buffer, commonly used to evaluate NTPDase activity, and in a more physiological Ringer modified buffer. Although there were some minor variations, there were no major changes either in the enzymatic activity or in the profile of NTPDase inhibition between the two buffers. Except for MRS2179, all other antagonists significantly inhibited these ecto-ATPases; NTPDase3 being the most sensitive to inhibition and NTPDase8 the most resistant. Estimated IC(50) showed that human NTPDases were generally more sensitive to the P2 receptor antagonists tested than the corresponding mouse isoforms. NF279 and reactive blue 2 were the most potent inhibitors of NTPDases which almost completely abrogated their activity at the concentration of 100 microM. In conclusion, reactive blue 2, suramin, NF279 and NF449, at the concentrations commonly used to antagonize P2 receptors, inhibit the four major ecto-ATPases. This information may reveal useful for the interpretation of some pharmacological studies of P2 receptors. In addition, NF279 is a most potent non-selective NTPDase inhibitor. Although P2 receptor antagonists do not display a strict selectivity toward NTPDases, their IC(50) values may help to discriminate some of these enzymes.
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Affiliation(s)
- Mercedes N Munkonda
- Centre de Recherche en Rhumatologie et Immunologie, Centre Hospitalier Universitaire de Québec, Université Laval, Québec, QC, Canada
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Shen JB, Cronin C, Sonin D, Joshi BV, Gongora Nieto M, Harrison D, Jacobson KA, Liang BT. P2X purinergic receptor-mediated ionic current in cardiac myocytes of calsequestrin model of cardiomyopathy: implications for the treatment of heart failure. Am J Physiol Heart Circ Physiol 2007; 292:H1077-84. [PMID: 17040972 PMCID: PMC6252253 DOI: 10.1152/ajpheart.00515.2006] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
P2X purinergic receptors, activated by extracellular ATP, mediate a number of cardiac cellular effects and may be important under pathophysiological conditions. The objective of the present study was to characterize the P2X receptor-mediated ionic current and determine its role in heart failure using the calsequestrin (CSQ) model of cardiomyopathy. Membrane currents under voltage clamp were determined in myocytes from both wild-type (WT) and CSQ mice. The P2X agonist 2-methylthio-ATP (2-meSATP) induced an inward current that was greater in magnitude in CSQ than in WT ventricular cells. The novel agonist, MRS-2339, an N-methanocarba derivative of 2-chloro-AMP relatively resistant to nucleotidase, induced a current in the CSQ myocyte similar to that by 2-meSATP. When administered via a miniosmotic pump (Alzet), it significantly increased longevity compared with vehicle-injected mice (log rank test, P = 0.02). The improvement in survival was associated with decreases in the heart weight-to-body weight ratio and in cardiac myocyte cross-sectional area [MRS-2339-treated mice: 281 +/- 15.4 (SE) mum(2), n = 6 mice vs. vehicle-treated mice: 358 +/- 27.8 mum(2), n = 6 mice, P < 0.05]. MRS-2339 had no vasodilator effect in mouse aorta ring preparations, indicating that its salutary effect in heart failure is not because of any vascular unloading. The cardiac P2X current is upregulated in the CSQ heart failure myocytes. Chronic administration of a nucleotidase-resistant agonist confers a beneficial effect in the CSQ model of heart failure, apparently via an activation of the cardiac P2X receptor. Cardiac P2X receptors represent a novel and potentially important therapeutic target for the treatment of heart failure.
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MESH Headings
- Adenine Nucleotides/pharmacology
- Adenine Nucleotides/therapeutic use
- Adenosine Monophosphate/analogs & derivatives
- Adenosine Monophosphate/pharmacology
- Adenosine Triphosphate/analogs & derivatives
- Adenosine Triphosphate/pharmacology
- Animals
- Benzenesulfonates/pharmacology
- Calsequestrin/genetics
- Calsequestrin/metabolism
- Cardiac Output, Low/etiology
- Cardiac Output, Low/prevention & control
- Cardiomyopathies/complications
- Cardiomyopathies/drug therapy
- Cardiomyopathies/metabolism
- Cardiomyopathies/pathology
- Disease Models, Animal
- Disease Progression
- Membrane Potentials/drug effects
- Mice
- Mice, Transgenic
- Myocytes, Cardiac/drug effects
- Myocytes, Cardiac/metabolism
- Myocytes, Cardiac/pathology
- Patch-Clamp Techniques
- Purinergic P2 Receptor Agonists
- Pyridoxal Phosphate/analogs & derivatives
- Pyridoxal Phosphate/pharmacology
- Receptors, Purinergic P2/drug effects
- Receptors, Purinergic P2/metabolism
- Receptors, Purinergic P2X4
- Thionucleotides/pharmacology
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Affiliation(s)
- Jian-Bing Shen
- Pat and Jim Calhoun Cardiology Center, University of Connecticut Health Center, 263 Farmington Ave., Farmington, CT 06030, USA.
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Ikeda M. Characterization of functional P2X(1) receptors in mouse megakaryocytes. Thromb Res 2006; 119:343-53. [PMID: 16626790 DOI: 10.1016/j.thromres.2006.03.007] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/30/2005] [Revised: 02/28/2006] [Accepted: 03/13/2006] [Indexed: 11/22/2022]
Abstract
INTRODUCTION Although accumulating evidence within the past 5 years strongly supports the importance of platelet P2X(1) receptors in hemostasis and thrombosis, P2X(1) receptors of platelet and/or its progenitor cell, megakaryocyte, have not been fully characterized. The aim of this study was to electrophysiologically and pharmacologically characterize the functional P2X(1) receptors on mouse megakaryocytes. MATERIALS AND METHODS The currents in response to nucleotides were examined using the patch-clamp whole-cell recording. RESULTS The agonist profile of megakaryocyte P2X(1) receptors was ATP>alpha,beta-methylene ATP>beta,gamma-methylene ATP. The P2X(1) receptors exhibited substantial monovalent as well as divalent cation permeability and the ratios of Na(+) to Cs(+) and Ca(2+) to Cs(+) permeability were 1 and 2.5, respectively. P2X receptor antagonists except suramin significantly inhibited the P2X(1) responses with an IC(50) values of 0.4 microM for pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (PPADS), 0.3 microM for 2',3'-O-(2,4,6-trinitophenyl)-adenosine 5'-triphosphate (TNP-ATP), 20 microM for reactive blue 2 (RB2), or 160 microM for 8,8'-(carbonylbis(imino-3,1-phenylene carbonylimino)bis(1,3,5-naphthalenetrisulfonic acid) (NF023), respectively. Suramin had no significant effect on the P2X(1) responses. In rat megakaryocytes, suramin similarly had no significant effect on the P2X(1) responses, but abolished the P2Y receptor-mediated responses, indicating that the suramin was active under present experimental condition. CONCLUSIONS These results provide the basic properties of mouse megakaryocyte P2X(1) receptors and would be helpful to examine the P2 receptor signaling in platelets and megakaryocytes.
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Affiliation(s)
- Masahiro Ikeda
- Department of Veterinary Pharmacology, Faculty of Agriculture, University of Miyazaki, Gakuenkibanadai-nishi 1-1, Miyazaki 889-2192, Japan
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González FA, Weisman GA, Erb L, Seye CI, Sun GY, Velázquez B, Hernández-Pérez M, Chorna NE. Mechanisms for inhibition of P2 receptors signaling in neural cells. Mol Neurobiol 2006; 31:65-79. [PMID: 15953812 DOI: 10.1385/mn:31:1-3:065] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/21/2004] [Accepted: 11/15/2004] [Indexed: 12/24/2022]
Abstract
Trophic factors are required to ensure neuronal viability and regeneration after neural injury. Although abundant information is available on the factors that cause the activation of astrocytes, little is known about the molecular mechanisms underlying the regulation of this process. Nucleotides released into the extracellular space from injured or dying neural cells can activate astrocytes via P2 nucleotide receptors. After a brief historical review and update of novel P2 receptor antagonists, this article focuses on recent advancements toward understanding molecular mechanisms that regulate G protein-coupled P2Y receptor signaling. Among P2Y receptor subtypes, the heptahelical P2Y2 nucleotide receptor interacts with vitronectin receptors via an RGD sequence in the first extracellular loop, and this interaction is required for effective signal transduction to activate mitogen-activated protein kinases ERK1/2, to mobilize intracellular calcium stores via activation of phospholipase C, protein kinase C isoforms, and to activate focal adhesion kinase and other signaling events. Ligation of vitronectin receptors with specific antibodies caused an inhibition of P2Y2 receptor-induced ERK1/2 and p38 phosphorylation and P2Y2 receptor-induced cytoskeleton rearrangement and DNA synthesis. Structure-function studies have identified agonist-induced phosphorylation of the C-terminus of the P2Y2 receptor, an important mechanism for receptor desensitization. Understanding selective mechanisms for regulating P2Y2 receptor signaling could provide novel targets for therapeutic strategies in the management of brain injury, synaptogenesis, and neurological disorders.
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Affiliation(s)
- Fernando A González
- Department of Chemistry, University of Puerto Rico, Río Piedras Campus, Puerto Rico.
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King BF, Liu M, Townsend-Nicholson A, Pfister J, Padilla F, Ford AP, Gever JR, Oglesby IB, Schorge S, Burnstock G. Antagonism of ATP responses at P2X receptor subtypes by the pH indicator dye, Phenol red. Br J Pharmacol 2006; 145:313-22. [PMID: 15778739 PMCID: PMC1576146 DOI: 10.1038/sj.bjp.0706187] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022] Open
Abstract
1 Many types of culture media contain a pH-sensitive dye. One commonly occurring dye, Phenol red sodium (Na(+)) salt, was tested for blocking activity at rat P2X(1-4) receptors (P2X(1-4)Rs) expressed in Xenopus oocytes. 2 Phenol red Na(+)-salt antagonised adenosine 5'-triphosphate (ATP) responses at P2X(1)R (IC(50), 3 microM) and, at higher concentrations, also blocked P2X(2)R and P2X(3)R. Phenol red Na(+)-salt, purified of lipophilic contaminants, blocked P2X(1)R and P2X(3)R by acting as an insurmountable antagonist. 3 Two lipophilic extracts of Phenol red antagonised ATP responses at P2XRs. Extract A was a potent antagonist at P2X(1)R (IC(50), 1.4 microM), whereas extract B was a potent antagonist at P2X(3)R (IC(50), 4.1 microM). A bisphenolic compound (RS151030) found in these extracts was a potent antagonist at P2X(1)R (IC(50), 0.3 microM) and at P2X(3)R (IC(50), 2.4 microM). 4 Phenolphthalein base was a potent irreversible antagonist at P2X(1)R (IC(50), 1 microM), whereas Phenolphthalein K(+)-salt was 25-fold less potent here. 5 Phenolphthalein base was a reversible antagonist of ATP responses at rat P2X(4)R (IC(50), 26 microM), whereas Phenolphthalein K(+)-salt was inactive. 6 Dimethyl sulphoxide (DMSO), used to dissolve lipophilic extracts, showed pharmacological activity by itself at rat P2X(1)R and P2X(4)R. 7 Thus, Phenol red and related compounds are antagonists at rat P2X(1)R, but are also active at other rat P2XRs. Phenolphthalein base is a newly identified, low potency antagonist of ATP responses at P2X(4)R. Culture media containing these red dyes should be used cautiously in future pharmacological studies of P2XRs. Also, wherever possible, the solvent DMSO should be used with caution.
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Affiliation(s)
- Brian F King
- University College London, Department of Physiology, Royal Free Campus, Rowland Hill Street, Hampstead, London NW3 2PF, UK.
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Jacobson KA, Mamedova L, Joshi BV, Besada P, Costanzi S. Molecular recognition at adenine nucleotide (P2) receptors in platelets. Semin Thromb Hemost 2005; 31:205-16. [PMID: 15852224 PMCID: PMC4423562 DOI: 10.1055/s-2005-869526] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/25/2022]
Abstract
Transmembrane signaling through P2Y receptors for extracellular nucleotides controls a diverse array of cellular processes, including thrombosis. Selective agonists and antagonists of the two P2Y receptors present on the platelet surface-the G (q)-coupled P2Y (1) subtype and the G (i)-coupled P2Y (12) subtype-are now known. High-affinity antagonists of each have been developed from nucleotide structures. The (N)-methanocarba bisphosphate derivatives MRS2279 and MRS2500 are potent and selective P2Y (1) receptor antagonists. The carbocyclic nucleoside AZD6140 is an uncharged, orally active P2Y (12) receptor antagonist of nM affinity. Another nucleotide receptor on the platelet surface, the P2X (1) receptor, the activation of which may also be proaggregatory, especially under conditions of high shear stress, has high-affinity ligands, although high selectivity has not yet been achieved. Although alpha,beta-methylene-adenosine triphosphate (ATP) is the classic agonist for the P2X (1) receptor, where it causes rapid desensitization, the agonist BzATP is among the most potent in activating this subtype. The aromatic sulfonates NF279 and NF449 are potent antagonists of the P2X (1) receptor. The structures of the two platelet P2Y receptors have been modeled, based on a rhodopsin template, to explain the basis for nucleotide recognition within the putative transmembrane binding sites. The P2Y (1) receptor model, especially, has been exploited in the design and optimization of antagonists targeted to interact selectively with that subtype.
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Affiliation(s)
- Kenneth A Jacobson
- Laboratory of Bioorganic Chemistry, National Institute of Diabetes & Digestive & Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0810, USA.
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Horner S, Menke K, Hildebrandt C, Kassack MU, Nickel P, Ullmann H, Mahaut-Smith MP, Lambrecht G. The novel suramin analogue NF864 selectively blocks P2X1 receptors in human platelets with potency in the low nanomolar range. Naunyn Schmiedebergs Arch Pharmacol 2005; 372:1-13. [PMID: 16158305 DOI: 10.1007/s00210-005-1085-z] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/18/2005] [Accepted: 07/14/2005] [Indexed: 11/28/2022]
Abstract
The role of ATP-stimulated P2X1 receptors in human platelets is still unclear. They may act alone or in synergy with other pathways, such as P2Y1 or P2Y12 receptors, to accelerate and enhance calcium mobilisation, shape change and aggregation. To date very few pharmacological means of selectively inhibiting platelet P2X1 receptors have been described, although recent work has shown that suramin is a useful lead compound for the development of high-affinity P2X1 antagonists. We therefore investigated the effects of a series of bivalent and tetravalent suramin analogues on alphabeta meATP (P2X1 receptors)-induced or ADP (P2Y1 receptors)-induced intracellular calcium increases and shape change, as well as on ADP-induced aggregation (P2Y1 & P2Y12 receptors) in human platelets. Changes in intracellular calcium were measured using standard fluorescence techniques, while shape change and aggregation were determined by turbidimetry. The novel tetravalent compound NF864 (8,8',8'',8'''-(carbonylbis(imino-5,1,3-benzenetriyl-bis(carbonylimino)))tetrakis-naphthalene-1,3,5-trisulfonic acid-dodecasodium salt) proved to be the most potent platelet P2X1 antagonist reported to date, blocking alphabeta meATP-induced Ca2+ increases and shape change in a concentration-dependent manner, with a pA2 of 8.17 and 8.49, respectively. The ability to inhibit the platelet P2X1 receptor displayed the following order : NF864 > NF449 > or = NF110 > NF023 = MK-HU1 = suramin. A different antagonistic profile was observed for ADP-induced Ca2+ increases, shape change and aggregation; however, overall four compounds showed sufficient ability to selectively inhibit P2X1 responses, with the order NF110 > NF449 > or = NF864 > or = MK-HU1. Therefore, these compounds should prove useful tools for investigating the functional significance of platelet P2X1 receptors in thrombosis and haemostasis, NF864 being the most promising compound.
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Affiliation(s)
- Susanne Horner
- Department of Pharmacology, University of Frankfurt, Marie-Curie-Strasse 9, 60439, Frankfurt (Main), Germany
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Glänzel M, Bültmann R, Starke K, Frahm AW. Structure-activity relationships of novel P2-receptor antagonists structurally related to Reactive Blue 2. Eur J Med Chem 2005; 40:1262-76. [PMID: 16153748 DOI: 10.1016/j.ejmech.2005.07.007] [Citation(s) in RCA: 33] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/10/2005] [Revised: 07/11/2005] [Accepted: 07/18/2005] [Indexed: 10/25/2022]
Abstract
P2 membrane receptors for nucleotides represent significant targets for experimental pharmacology and drug research. In earlier publications, we have shown that Reactive Blue 2 (RB 2), one of the most widely used P2-receptor antagonists, displays only moderate affinity and does not discriminate between native P2X- and P2Y-receptor subtypes. In the present study we have pharmacologically evaluated a series of 15 synthesized and re-evaluated four commercially obtained and chromatographically purified RB 2 type anthraquinone derivatives on contractions of the rat vas deferens (RVD) elicited by alpha,beta-methylene ATP (alpha,beta-meATP), mediated by P2X1-receptors, and relaxations of the carbachol-precontracted guinea-pig taenia coli (GPTC) elicited by adenosine 5'-O-(2-thiodiphosphate) (ADPbetaS), mediated by P2Y1-like receptors. Based on the structure-activity relationships (SAR) it is concluded that hydrophobic interactions of aromatic pi-electron systems, hydrogen bonds with nitrogen as donor and acceptor atoms, and, particularly, position, conformational distance and number of anionic sulfonate groups are of great importance for the blockade of the two native P2-receptor subtypes. We have also identified novel, for the most part reversible antagonists that bind with higher affinity and improved subtype selectivity in comparison to RB 2. In particular, 1-amino-4-{4-[4-chloro-6-(2-sulfonatophenylamino)-[1,3,5]triazine-2-ylamino]-2-sulfonatophenylamino}-9,10-dioxo-9,10-dihydroanthracene-2-sulfonic acid trisodium salt (MG 50-3-1) is the most potent antagonist at the P2Y1-like-receptors of the GPTC reported so far (IC50=4.6 nM). It is significantly less potent as reversible antagonist at the P2X1-receptors of the RVD (IC50=2.8 microM). Thus, MG 50-3-1 represents a selective pharmacological tool and may be a lead compound for future investigations.
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Affiliation(s)
- Markus Glänzel
- Institut für Pharmazeutische Wissenschaften Albertstrasse 25, D-79104 Freiburg i.Br., Germany
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Hechler B, Magnenat S, Zighetti ML, Kassack MU, Ullmann H, Cazenave JP, Evans R, Cattaneo M, Gachet C. Inhibition of platelet functions and thrombosis through selective or nonselective inhibition of the platelet P2 receptors with increasing doses of NF449 [4,4',4'',4'''-(carbonylbis(imino-5,1,3-benzenetriylbis-(carbonylimino)))tetrakis-benzene-1,3-disulfonic acid octasodium salt]. J Pharmacol Exp Ther 2005; 314:232-43. [PMID: 15792995 DOI: 10.1124/jpet.105.084673] [Citation(s) in RCA: 69] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
Our aim was to determine whether the newly described P2X1 antagonist NF449 [4,4',4'',4'''-(carbonylbis(imino-5,1,3-benzenetriylbis(carbonylimino)))tetrakis-benzene-1,3-disulfonic acid octasodium salt] could selectively antagonize the platelet P2X1 receptor and how it affected platelet function. NF449 inhibited alpha,beta-methyleneadenosine 5'-triphosphate-induced shape change (IC50 = 83 +/- 13 nM; n = 3) and calcium influx (pA2 = 7.2 +/- 0.1; n = 3) (pIC50 = 6.95) in washed human platelets treated with apyrase to prevent desensitization of the P2X1 receptor. NF449 also antagonized the calcium rise mediated by the P2Y1 receptor, but with lower potency (IC50 = 5.8 +/- 2.2 microM; n = 3). In contrast, it was a very weak antagonist of the P2Y12-mediated inhibition of adenylyl cyclase activity. Selective blockade of the P2X1 receptor with NF449 led to reduced collagen-induced aggregation, confirming a role of this receptor in platelet activation induced by collagen. Intravenous injection of 10 mg/kg NF449 into mice resulted in selective inhibition of the P2X1 receptor and decreased intravascular platelet aggregation in a model of systemic thromboembolism (35 +/- 4 versus 51 +/- 3%) (P = 0.0061; n = 10) but without prolongation of the bleeding time (106 +/- 16 versus 78 +/- 7 s; n = 10) (N.S.; P = 0.1209). At a higher dose (50 mg/kg), NF449 inhibited the three platelet P2 receptors. This led to a further reduction in platelet consumption compared with mice injected with saline (13 +/- 4 versus 42 +/- 3%) (P = 0.0002; n = 5). NF449 also reduced dose-dependently the size of thrombi formed after laser-induced injury of mesenteric arterioles. Overall, our results indicate that NF449 constitutes a new tool to investigate the functions of the P2X1 receptor and could be a starting compound in the search for new antithrombotic drugs targeting the platelet P2 receptors.
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Affiliation(s)
- Béatrice Hechler
- INSERM U. 311, Etablissement Français du Sang-Alsace, 10, rue Spielmann, Strasbourg Cédex, France
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