DOCSLIB.ORG

Involvement of a P2X7 Receptor in the Acrosome Reaction Induced By

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Involvement of a P2X7 Receptor in the Acrosome Reaction Induced By ORIGINAL RESEARCH ARTICLE 3068 JournalJournal ofof Cellular Involvement of a P2X7 Receptor Physiology in the Acrosome Reaction Induced by ATP in Rat Spermatozoa JORGE L. TORRES-FUENTES, MARIANA RIOS, AND RICARDO D. MORENO* Departamento de Fisiologıa, Facultad de Ciencias Biologicas, Pontificia Universidad Catolica de Chile, Santiago, Chile The acrosome reaction (AR) is the exocytosis of the acrosomal vesicle in response to different physiological and non-physiological stimuli. Particularly in mammals, the AR is needed for sperm to fuse with the oocyte plasma membrane, and it occurs only in capacitated sperm. Previous evidence in the literature indicates that extracellular ATP induces the AR in capacitated human and bovine spermatozoa, but its receptor has not yet been identified. The aim of this work was to define a putative ATP receptor in rat spermatozoa using pharmacological and biochemical approaches. We found that ATP induced the AR only in capacitated rat spermatozoa, which was inhibited in the presence of two general inhibitors of ATP receptors (P2 receptors), Suramin, and oxidized ATP (oATP), and one inhibitor of P2X receptor (pyridoxalphosphate-6-azophenyl-20,40-disulfonic acid [PPADS]). In addition, the AR induced by ATP in capacitated rat spermatozoa was inhibited by brilliant blue-G (BB-G) and 17-b-oestradiol, two blockers of P2X7 receptors. Moreover, the ATP analog 20 (30)-O-(4-benzoylbenzoyl) ATP (BzATP) was almost 500 times more potent than ATP to induce the AR, which agrees with the pharmacology of a P2X7 receptor. Here, we show the presence of P2X7 receptor by Western blot and its localization in the tail and acrosome by indirect immunofluorescence. Finally, we quantify the presence of ATP in the rat oviduct during the estrous cycle. We found that the ATP concentration within the lumen of the oviduct is similar to those required to induce acrosome reaction, which agree with its role during in vivo fertilization. Therefore, our results strongly suggest that ATP induces the AR in capacitated rat spermatozoa through a P2X7 receptor, which may be functional during in vivo fertilization. J. Cell. Physiol. 230: 3068–3075, 2015. © 2015 Wiley Periodicals, Inc. It is known that mammalian ejaculated sperm are not capable of (P2X1, P2X2, P2X3, P2X4, P2X5, and P2X7) or heteromeric fertilizing eggs when they leave the male reproductive tract (P2X2/3 and P2X1/5). They are differentially expressed in (Barros et al., 1996; Yanagimachi, 2011) because they need to different tissues and throughout the entire male reproductive undergo a series of biochemical changes known as tract (Burnstock, 2013). The presence of different P2X “capacitation,” before they bind and fuse with the oocyte receptors can be determined by the use of specific antibodies plasma membrane (Austin, 1951; Chang, 1951). These changes or different agonist or antagonists such as occur during female genital tract transit and can be mimicked in pyridoxalphosphate-6-azophenyl-20,40-disulfonic acid vitro in a chemically defined culture medium or capacitating (PPADS), 20,30-O-(2,4,6-trinitrophenyl)-ATP (oATP), Suramin medium (CM; Visconti et al., 1995a,b; Salicioni et al., 2007). The or Coomassie blue brillant-G (North, 2002). endpoint of capacitation is the acrosome reaction (AR), which Extracellular ATP induces AR at concentrations above is the exocitosis of the acrosomal vesicle localized over the 500 mM in humans (Foresta et al., 1992) and bovine sperm head (Barros et al., 1967; Ramalho-Santos et al., 2002; spermatozoa (Luria et al., 2002). One report shows that Moreno and Alvarado, 2006; Zanetti and Mayorga, 2009). The extracellular ATP activate P2X2 receptors in non-capacitated acrosome is an acid compartment derived from Golgi vesicles mouse spermatozoa causing a large and fast-activating inward and lysosomes containing hydrolytic enzymes that supposedly current (Navarro et al., 2011). However, genetic ablation of help the sperm to cross the zona pellucida (Barros et al., 1992; P2X2 receptor does not have any consequence in fertilization, Moreno, 2003; Codelia et al., 2005; Moreno and Alvarado, suggesting the participation of other P2 receptors. In addition, 2006; Yanagimachi, 2011). In addition, the AR changes the ATP can induce intracellular calcium to increase in a post-acrosomal membrane in order to prepare the sperm to concentration-dependent manner in the mouse head fuse with the egg’s plasma membrane (Barros et al., 1992; Inoue et al., 2005; Moreno et al., 2011) During the AR, there is a massive increase in the intracellular free calcium that triggers the membrane fusion machinery between the outer acrosomal membrane and the adjacent Contract grant sponsor: FONDECYT; plasma membrane (Darszon et al., 2005). In addition, the AR Contract grant number: 1110778. dissipates the acid intra-acrosomal pH, and soluble proteins *Correspondence to: Ricardo D. Moreno, Departamento de and active proteases are released to the extracellular milieu Fisiologıa, Facultad de Ciencias Biologicas, Pontificia Universidad (Barros et al., 1992; Codelia et al., 2005; Buffone et al., 2009a,b). Catolica de Chile, Alameda 340, Santiago, Chile. However, recent experiments have cast doubt about the real E-mail: [email protected] physiological role of the zona pellucida as the inducer of the AR Manuscript Received: 27 December 2014 (Hirohashi et al., 2011; Inoue et al., 2011). Manuscript Accepted: 11 May 2015 Purinergic P2X receptors are transmembrane ion channels Accepted manuscript online in Wiley Online Library that open in response to the binding of extracellular ATP and (wileyonlinelibrary.com): 18 May 2015. elicit different physiological responses. Channels form as DOI: 10.1002/jcp.25044 multimers of several subunits, which could be homomeric © 2015 WILEY PERIODICALS, INC. INVOLVEMENT OF A P2X7 RECEPTOR IN THE ACROSOME REACTION 3069 independent of CATSPER channel (Xia and Ren, 2009), The acrosome reaction was evaluated by the Coomassie blue suggesting that other channels could also be involved in the AR. dye technique (Bendahmane et al., 2002; Cisternas and Moreno, Giving the previous antecedents, the major goal of this work 2006). In brief, fixed spermatozoa were washed three times with was to a characterize a putative ATP receptor involved in the 100 mM ammonium acetate by centrifugation at 3,000 rpm, layered AR in rat spermatozoa. into microscope glass slides and dried at 30°C. Slides were washed in methanol, distilled water and methanol for 5 min at room Materials and Methods temperature, submerged in Coomassie brilliant blue G-250 Animals solution for 2 min, washed with distilled water and mounted with Entellan (Merck). All samples were observed in a phase contrast Adult male Sprague–Dawley rats were acquired from the Animal light microscope with objective Â40 (Olympus CX31, Tokyo, Facility of our institution. The rats were housed under a 12L:12D Japan) and at least 200 cells were counted for acrosome negative cycle with water and rat chow ad libitum and they were killed by or positive staining from at least three different rats. high CO2 atmosphere. Investigations were conducted in accordance with the rules laid down by the Consortium for Indirect inmunofluorescence Developing a Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching and by the National Research Rat spermatozoa were extracted in NCM and fixed as indicate Council. All animal protocols were endorsed by the Chilean above. Fixed samples were washed with PBS by centrifugation at National Fund of Science and Technology (FONDECYT). 3,000 rpm for 5 min, layered into microscope glass slides and dried at 30°C. Slides were washed twice with PBS, and cells were Reagents permeabilized with Triton X-100 1%-PBS for 10 min. Non-specific reaction sites were blocked with 3% BSA-PBS for 0 Adenosine-5 -triphosphoric acid disodium salt (ATP) was 1 h and incubated overnight with a rabbit anti-P2RX7 (dilution purchased from Merck (Darmstadt, Germany), adenosine 1:200) antibody at 4°C in a humidified chamber. Then, samples 1 50-triphosphate, periodate oxidized sodium salt (oATP), and were washed three times in PBS, incubated with Alexa Fluor suramin sodium (Suramin) were purchased from Santa Cruz 488 donkey anti-rabbit antibody for 3 h, washed three times and Biotechnology (Santa Cruz, CA), 20(30)-O-(4-benzoylbenzoyl) incubated with 1 mM PI/0.1%Tween-PBS for 15 min at room adenosine-50-triphosphate triethylammonium salt (BzATP), and temperature in a humidified chamber protected from light. rabbit anti-P2RX7 antibody (ab77413) were obtained from Abcam Finally, samples were washed and mounted in Fluoromount (Cambridge, England); goat anti-rabbit IgG HRP and anti-mouse mounting medium (Sigma) and observed under a Bx51 IgG (H þ L) HRP antibodies were acquired from KPL fluorescence inverted microscopy (Olympus, Center Valley, PA) (Gaithersburg, MD); mouse anti-b-tubulin antibody (32-2600), and photographed with a PM-30 digital camera (Olympus). 1 Alexa Fluor 488 donkey anti-rabbit antibody (A-21206), and Propidium iodide (PI) were acquired from Life technologies Spermatozoa protein extraction and Western blotting (Carlsbad, CA); pyridoxalphosphate-6-azophenyl-20,40-disulfonic acid (PPADS), Coomassie brilliant blue-G (BB-G) (B0770), Sperm protein extraction was performed by homogenizing sperm 17b-estradiol (E8875) (17b-E2), BSA, and other reagents were suspensions in a buffer containing 1 M NaCl, 1 mM EDTA, purchased from Sigma (St. Louis, MO). 10 mg/ml PMSF, 1% Triton X-100, 20 mM Tris–HCl pH 7.4, plus a protease inhibitor cocktail (Sigma) including 2 mM AEBSF fl Rat sperm capacitation [4-(2-Aminoethyl) benzenesulfonyl uoride hydrochloride], 0.3 mM aprotinin, 130 mM bestatin hydrochloride, 14 mME-64, Caudal epididymal sperms were collected from 4-month-old male 1 mM EDTA, 1 mM leupeptin hemisulfatein then centrifuged for Sprague–Dawley rats. Cauda epididymis of each animal was placed 10 min at 10,000g at 4°C. Proteins were resolved on a 7% into 3 ml of non-capacitating medium (NCM), containing 86.8 mM polyacrylamide gel (SDS–PAGE) under reducing and denaturing NaCl, 4.17 mM KCl, 3.5 mM CaCl2 Á 2H2O, 1.22 mM conditions then transferred to nitrocellulose at 400 mA for 2 h.
Load more

Recommended publications
  • Expression of Purinergic Receptors on Microglia in the Animal Model Of
    www.nature.com/scientificreports OPEN Expression of purinergic receptors on microglia in the animal model of choroidal neovascularisation Lu Li1*, Juejun Liu1, Amin Xu1, Peter Heiduschka2, Nicole Eter2 & Changzheng Chen1 To investigate the efect of P2 receptor on microglia and its inhibitor PPADS on choroidal neovascularization. Forty CX3CR1GFP/+ mice were randomly divided into 8 groups. In addition to the normal group, the rest of groups were receiving laser treatment. The retina and choroid from the second, third, fourth and ffth group of mice were taken in the 1, 4, 7, 14 days after laser treatment. The mice in the sixth and seventh group received intravitreal injection of 2 µl PPADS or PBS respectively immediately after laser treatment. The mice in the eighth group received topical application of PPADS once per day of three days. The mice in sixth, seventh and eighth group received AF and FFA examination on the fourth day after laser treatment. Immunofuorescence histochemical staining and real-time quantitative PCR were used to evaluate P2 expression and its efect on choroidal neovascularization. After laser treatment, activated microglia can express P2 receptors (P2X4, P2X7, P2Y2 and P2Y12). The expression of P2 increased on the frst day after laser damage, peaked on the fourth day (tP2X4 = 6.05, tP2X7 = 2.95, tP2Y2 = 3.67, tP2Y12 = 5.98, all P < 0.01), and then decreased. After PPADS inhibition, compared with the PBS injection group, the mRNA of P2X4, P2X7, P2Y2 and P2Y12 were decreased signifcantly in the PPADS injection group (tP2X4 = 5.54, tP2X7 = 9.82, tP2Y2 = 3.86, tP2Y12 = 7.91, all P < 0.01) and the PPADS topical application group (tP2X4 = 3.24, tP2X7 = 5.89, tP2Y2 = 6.75, tP2Y12 = 4.97, all P < 0.01).
    [Show full text]
  • The Inflammasome Promotes Adverse Cardiac Remodeling Following Acute
    The inflammasome promotes adverse cardiac remodeling following acute myocardial infarction in the mouse Eleonora Mezzaromaa,b,c,1, Stefano Toldoa,b,1, Daniela Farkasb, Ignacio M. Seropiana,b,c, Benjamin W. Van Tassellb,c, Fadi N. Sallouma, Harsha R. Kannana,b, Angela C. Mennaa,b, Norbert F. Voelkela,b, and Antonio Abbatea,b,2 aVCU Pauley Heart Center, bVCU Victoria Johnson Center, and cSchool of Pharmacy, Virginia Commonwealth University, Richmond, VA 23298 Edited* by Charles A. Dinarello, University of Colorado Denver, Aurora, CO, and approved October 19, 2011 (received for review May 31, 2011) Acute myocardial infarction (AMI) initiates an intense inflamma- and increased caspase-1–mediated cell death in a more severe tory response that promotes cardiac dysfunction, cell death, and model of ischemia without reperfusion. We also describe phar- ventricular remodeling. The molecular events underlying this macologic inhibition of cryopyrin and P2X7 to prevent inflam- inflammatory response, however, are incompletely understood. masome formation and ameliorate cardiac damage as a potential In experimental models of sterile inflammation, ATP released from basis for translational investigation. dying cells triggers, through activation of the purinergic P2X7 receptor, the formation of the inflammasome, a multiprotein Results complex necessary for caspase-1 activation and amplification of Caspase-1 Is Activated in AMI. Caspase-1 mRNA synthesis in- the inflammatory response. Here we describe the presence of the creased severalfold in the heart at 3 and 7 d after AMI (Fig. S1). inflammasome in the heart in an experimental mouse model of Caspase-1 activation was also increased at 7 d as measured by AMI as evidenced by increased caspase-1 activity and cytoplasmic increased procaspase-1, increased cleaved caspase-1, and in- aggregates of the three components of the inflammasome—apo- creased cleaved/procaspase-1 ratio (Fig.
    [Show full text]
  • Ion Channels
    UC Davis UC Davis Previously Published Works Title THE CONCISE GUIDE TO PHARMACOLOGY 2019/20: Ion channels. Permalink https://escholarship.org/uc/item/1442g5hg Journal British journal of pharmacology, 176 Suppl 1(S1) ISSN 0007-1188 Authors Alexander, Stephen PH Mathie, Alistair Peters, John A et al. Publication Date 2019-12-01 DOI 10.1111/bph.14749 License https://creativecommons.org/licenses/by/4.0/ 4.0 Peer reviewed eScholarship.org Powered by the California Digital Library University of California S.P.H. Alexander et al. The Concise Guide to PHARMACOLOGY 2019/20: Ion channels. British Journal of Pharmacology (2019) 176, S142–S228 THE CONCISE GUIDE TO PHARMACOLOGY 2019/20: Ion channels Stephen PH Alexander1 , Alistair Mathie2 ,JohnAPeters3 , Emma L Veale2 , Jörg Striessnig4 , Eamonn Kelly5, Jane F Armstrong6 , Elena Faccenda6 ,SimonDHarding6 ,AdamJPawson6 , Joanna L Sharman6 , Christopher Southan6 , Jamie A Davies6 and CGTP Collaborators 1School of Life Sciences, University of Nottingham Medical School, Nottingham, NG7 2UH, UK 2Medway School of Pharmacy, The Universities of Greenwich and Kent at Medway, Anson Building, Central Avenue, Chatham Maritime, Chatham, Kent, ME4 4TB, UK 3Neuroscience Division, Medical Education Institute, Ninewells Hospital and Medical School, University of Dundee, Dundee, DD1 9SY, UK 4Pharmacology and Toxicology, Institute of Pharmacy, University of Innsbruck, A-6020 Innsbruck, Austria 5School of Physiology, Pharmacology and Neuroscience, University of Bristol, Bristol, BS8 1TD, UK 6Centre for Discovery Brain Science, University of Edinburgh, Edinburgh, EH8 9XD, UK Abstract The Concise Guide to PHARMACOLOGY 2019/20 is the fourth in this series of biennial publications. The Concise Guide provides concise overviews of the key properties of nearly 1800 human drug targets with an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties.
    [Show full text]
  • Suramin As a Chemo- and Radio-Sensitizer
    SURAMIN AS A CHEMO- AND RADIO-SENSITIZER: PRECLINICAL TRANSLATIONAL STUDIES DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Yan Xin, M.S. ***** The Ohio State University 2006 Dissertation Committee: Dr. M. Guillaume Wientjes, Adviser Approved by Dr. Jessie L.-S. Au ____________________________________ Adviser Dr. Duxin Sun Graduate Program in Pharmacy ABSTRACT Previous studies in our laboratory showed that low-dose suramin, an inhibitor of fibroblast growth factor action, enhances sensitivity of various human tumors in preclinical and clinical studies to chemotherapy. Chemosensitization required apoptosis, and increased the extent and duration of the induction of apoptosis. The primary focus of this dissertation research was to explore, in preclinical studies, therapeutic approaches for therapy enhancement based on this mechanism. A phase III clinical trial in superficial bladder cancer, which emanated from our laboratory, showed that optimizing mitomycin C delivery nearly doubled the recurrence- free survival of treated patients to 40%. Tumor sensitization with suramin might yield further therapeutic improvements, and was investigated in in vitro and in vivo studies. Studies in Chapter 2 demonstrated enhanced antitumor activity of mitomycin C, administered at subtherapeutic and therapeutic regimens. Various preclinical and clinical studies determined that suramin sensitized tumor tissue at low but not at high concentrations, presumably due to additional pharmacologic effects at elevated concentrations. Studies to overcome this limitation, especially in tumors containing high fibroblast growth factor concentrations, used pentosan polysulfate, another nonspecific FGF inhibitor. This agent was also a chemosensitizer, but combined use with suramin did not increase the overall efficacy (Chapter 3).
    [Show full text]
  • Antidepressant-Like Effects of P2 Purinergic Antagonist PPADS Is Dependent on Serotonergic and Noradrenergic Integrity
    bioRxiv preprint doi: https://doi.org/10.1101/086983; this version posted November 10, 2016. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Title: Antidepressant-like effects of P2 purinergic antagonist PPADS is dependent on serotonergic and noradrenergic integrity. Authors: Cassiano R. A. F. Diniza, Murilo Rodriguesb, Plínio C. Casarottoc, Vítor S. Pereirad, Carlos C. Crestanie, Sâmia R.L. Jocab,d*. aSchool of Medicine, Campus USP, Ribeirão Preto, SP 14049-900, Brazil bDepartment of Physics and Chemistry, School of Pharmaceutical Sciences, Campus USP, Ribeirão Preto, SP 14040-904, Brazil. cNeuroscience Center, University of Helsinki, Finland dDepartment of Clinical Medicine - Translational Neuropsychiatry Unit, Aarhus University, Denmark eLaboratory of Pharmacology, School of Pharmaceutical Sciences, UNESP - Universidade Estadual Paulista, Araraquara, SP, Brazil *Corresponding Author: Sâmia Joca. Department of Physics and Chemistry - School of Pharmaceutical Sciences of Ribeirão Preto (FCFRP) University of São Paulo (USP). AvCafe, s/n, 14040-903, Ribeirão Preto - SP, Brazil. Phone: +55-16-33154705 -Fax: +55-16-33154880. e-mail: [email protected] Samia R. L. Joca: [email protected] 1 bioRxiv preprint doi: https://doi.org/10.1101/086983; this version posted November 10, 2016. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
    [Show full text]
  • Brian F King and Andrea Townsend- Nicholson University College
    BrianFKingandAndreaTownsend- P2XReceptors Nicholson P2Xreceptors(P2XRs)areligand-gatedion-channels (LGICs)widelydistributedamongstvarioustissue UniversityCollegeLondon,GowerStreet, types,includingautonomic,central,entericand London,WC1E6BT,UK. sensoryneurons,cochlearandretinalcells, endotheliumandepithelium,vascularandvisceral BrianKingisaSeniorLecturerinthe smoothmuscle,heartanddevelopingskeletal DepartmentofPhysiology,andAndrea muscle,bone,haemopoieticcells.TheseLGICsserve Townsend-NicholsonaLecturerinthe tocontroltheexcitabilityoftheirhostcellsthrough DepartmentofBiochemistryandMolecular twoprocesses:i)influxofextracellularsodiumionsto elicitdepolarisation,andii)influxofextracellular Biology.Theyshareacommoninterestin calciumionstoactivateinternalenzymes. purinesignallingatrecombinantandnative P1andP2receptorsandhaveworked P2XRassembly togetheroverthelastsevenyearsonthe Sevenbuildingblocks, orsubunitproteins,are isolationandcharacterisationofnucleotide involvedintheconstructionofP2XRs.Theseseven receptors. buildingblocks–theP2X17toP2Xsubunits–have beenclonedfromhumanandratcDNAlibrariesand, insomecases,alsofrommouse,guinea-pig,chick andzebrafishlibraries.1,2 Fewstructuraldifferences havebeennotedbetweenspeciesorthologuesofthe Introduction sameP2Xsubunit.However,thedegreeofhomology canbeaslittleas27%betweendifferentsubunitsof Extracellularnucleotidesignallingmolecules,suchas thesamespecies.1 Furthermore,theP2Xgenesare ATP,ADP,UTPandUDP,workthroughspecific complexand,duringtranscription,canundergo ionotropic(P2X)ormetabotropic(P2Y)cell-surface
    [Show full text]
  • P2Y6 Receptor Antagonist MRS2578 Inhibits Neutrophil Activation and Aggregated Neutrophil Extracellular Trap Formation
    P2Y6 Receptor Antagonist MRS2578 Inhibits Neutrophil Activation and Aggregated Neutrophil Extracellular Trap Formation Induced by Gout-Associated Monosodium Downloaded from This information is current as Urate Crystals of January 6, 2017. Payel Sil, Craig P. Hayes, Barbara J. Reaves, Patrick Breen, Shannon Quinn, Jeremy Sokolove and Balázs Rada J Immunol 2017; 198:428-442; Prepublished online 30 http://www.jimmunol.org/ November 2016; doi: 10.4049/jimmunol.1600766 http://www.jimmunol.org/content/198/1/428 Supplementary http://www.jimmunol.org/content/suppl/2016/11/29/jimmunol.160076 Material 6.DCSupplemental.html at Texas A & M University Medical Sciences Library on January 6, 2017 References This article cites 120 articles, 32 of which you can access for free at: http://www.jimmunol.org/content/198/1/428.full#ref-list-1 Subscriptions Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscriptions Permissions Submit copyright permission requests at: http://www.aai.org/ji/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/cgi/alerts/etoc The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 9650 Rockville Pike, Bethesda, MD 20814-3994. Copyright © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology P2Y6 Receptor Antagonist MRS2578 Inhibits Neutrophil Activation and Aggregated Neutrophil Extracellular Trap Formation Induced by Gout-Associated Monosodium Urate Crystals Downloaded from Payel Sil,* Craig P. Hayes,* Barbara J. Reaves,* Patrick Breen,† Shannon Quinn,‡ Jeremy Sokolove,x,{ and Bala´zs Rada* Human neutrophils (polymorphonuclear leukocytes [PMNs]) generate inflammatory responses within the joints of gout patients upon encountering monosodium urate (MSU) crystals.
    [Show full text]
  • MRS Researchtools Full List 02-08-21
    Compounds introduced or investigated in Ken Jacobson’s lab ([email protected]) at NIDDK or through collaborations. Orange compounds are or will soon be available commercially as research tools: Abbreviation Action Reference (year, vol:page) ** AB-MECA selective A3 adenosine agonist, to radioiodinate JMC (1994) 37:636 ADAC selective A1 adenosine agonist BP (1987) 36:1697 Alexa488-APEC fluorescent A2A adenosine receptor agonist EJP (2008) 590:36 APEC selective A2A adenosine agonist* JMC (1994) 37:3614 3’-Benzylamino-3’-deoxy-ATP Potent P2X agonist (25) DDR (1994) 31:206 Benzyl-NECA potent A3 adenosine agonist MP (1994) 45:1101 BTH4 non-N-containing adenosine antagonist (7) JMC (1996) 39:398 Cl-IB-MECA selective A3 adenosine agonist JMC (1994) 37:3614 CMB6446 selective A2B adenosine antagonist BMC (2003) 11:77 CSC selective A2A adenosine antagonist JMC (1993) 36:1333 DBXR xanthine riboside as adenosine antagonist JMC (1994) 37:4020 DBXRM selective A3 adenosine agonist JMC (1994) 37:4020 m-DITC-ADAC selective A1 adenosine agonist affinity label (46) JMC (1989) 32:1043 p-DITC-ADAC selective A1 adenosine agonist affinity label (45) JMC (1989) 32:1043 m-DITC-APEC selective A2A adenosine agonist affinity label (11) JMR (1989) 32:1043 p-DITC-APEC selective A2A adenosine agonist affinity label (12) JMR (1989) 32:1043 m-DITC-XAC selective A1 adenosine antagonist affinity label (10) JMC (1989) 32:1043 p-DITC-XAC selective A1 adenosine antagonist affinity label (9) JMC (1989) 32:1043 DU124183 selective A3 adenosine allosteric enhancer MP (2003)
    [Show full text]
  • The Effects of Purine Compounds on the Isolated Aorta of the Frog Rana Temporaria Gillian E
    British Journal of Pharmacology (1996) 117, 873-878 B 1996 Stockton Press All rights reserved 0007-1188/96 $12.00 S The effects of purine compounds on the isolated aorta of the frog Rana temporaria Gillian E. Knight & 'Geoffrey Burnstock Department of Anatomy and Developmental Biology and Centre for Neuroscience, University College London, Gower Street, London WC1E 6BT 1 In the isolated aorta of the frog, Rana temporaria, adenosine concentration-dependently, endothelium-independently relaxed adrenaline pre-constricted vessels. None of the adenosine analogues including D-5'-(N-ethylcarboxamide) adenosine (NECA), R- and S-N6-(2-phenylisopropyl) adenosine (R- and S-PIA) and 2-chloroadenosine (2-CA), or the more selective Al, A2 and A3 agonists cyclopentyladenosine (CPA), CGS 21680 and Ml-(3-iodobenzyl) adenosine-5'-N-methylcarboxamide (IB-MECA) respectively, had any effect. 2 The non-selective adenosine antagonist, 8-p-sulphophenyl-theophylline (8-pSPT; 30 giM) failed to inhibit adenosine relaxations, as did NG-nitro-L-arginine methyl ester (L-NAME; 0.1 mM) and indomethacin (30 gM). 3 Adenosine 5'-triphosphate (ATP), ca,f,-methylene ATP (a,#-MeATP), ,B,y-methylene ATP (fl,y- MeATP), 2-methylthio ATP (2-MeSATP) and uridine 5'-triphosphate (UTP) all concentration- dependently contracted the frog aorta. ATP and a,,B-MeATP were equipotent and more potent than UTP and ,B,y-MeATP; 2-MeSATP had little activity. 4 The P2-purinoceptor antagonist, suramin (0.1 mM) inhibited contractions to x,,B-MeATP but not to ATP. Pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS; 30 gM) also inhibited contractions to a,,B-MeATP but not to ATP.
    [Show full text]
  • Review One Year in Review 2018: Gout
    Review One year in review 2018: gout L. Punzi1, A. Scanu2, P. Spinella3, P. Galozzi2, F. Oliviero2 1Centre for Gout and Metabolic Bone ABSTRACT In line with the editorial policy of this and Joint Diseases, Rheumatology, Gout is the most common form of inflam- journal to publish yearly updates on the SS Giovanni and Paolo, Venice, Italy matory arthropathy, and is associated most relevant topics of rheumatology, 2Rheumatology Unit, and 3Clinical with excruciating pain, major impair- we will provide here an overview of Nutrition Unit, Department of Medicine - DIMED, University of Padova, ltaly. ment of quality of life, and increased the recent literature on novel treatments risk of comorbidities and mortality. in gout (8-22). This article reviews the Leonardo Punzi, MD, PhD Anna Scanu, PhD Although gout has somehow been ne- new clinical and experimental evidence Paolo Spinella, MD glected by researchers and clinicians in about gout that emerged in 2017 and Paola Galozzi, PhD the past, in more recent times there has in the first half of 2018. Relevant pa- Francesca Oliviero, PhD been a renewed interest in this disease, pers published from January 2017 were Please address correspondence to: which has led to major improvements in identified by a PubMed search, last Prof. Leonardo Punzi, its management. update at the beginning of July 2018; Centre for Gout and Metabolic This article reviews the new clinical papers were then selected for inclusion Bone and Joint Diseases, and experimental evidence about gout according to the authors’ judgement. Padiglione Mendicanti I piano, that emerged in 2017 and in the first Ospedale Civile SS.
    [Show full text]
  • Activation of the NLRP3 Inflammasome in Dendritic Cells Induces IL-1Β–Dependent Adaptive Immunity Against Tumors
    A RTICLES Activation of the NLRP3 inflammasome in dendritic cells induces IL-1β–dependent adaptive immunity against tumors François Ghiringhelli1–4,18, Lionel Apetoh1,2,5,6,18, Antoine Tesniere2,5,7,18, Laetitia Aymeric1,2,5,18, Yuting Ma1,2,5, Carla Ortiz1,2,5,8, Karim Vermaelen1,2,5,9, Theocharis Panaretakis2,5,7, Grégoire Mignot1–4, Evelyn Ullrich1,2,5, Jean-Luc Perfettini2,5,7, Frédéric Schlemmer2,5,7, Ezgi Tasdemir2,5,7, Martin Uhl10, Pierre Génin11, Ahmet Civas11, Bernhard Ryffel12, Jean Kanellopoulos13, Jürg Tschopp14, Fabrice André1,2,5, Rosette Lidereau15, Nicole M McLaughlin16, Nicole M Haynes16, Mark J Smyth16,18, Guido Kroemer2,5,7,18 & Laurence Zitvogel1,2,5,17,18 The therapeutic efficacy of anticancer chemotherapies may depend on dendritic cells (DCs), which present antigens from dying cancer cells to prime tumor-specific interferon- (IFN-)–producing T lymphocytes. Here we show that dying tumor cells release ATP, which then acts on P2X7 purinergic receptors from DCs and triggers the NOD-like receptor family, pyrin domain containing-3 protein (NLRP3)-dependent caspase-1 activation complex (‘inflammasome’), allowing for the secretion of interleukin-1 (IL-1). The priming of IFN-–producing CD8+ T cells by dying tumor cells fails in the absence of a functional IL-1 receptor 1 and in Nlpr3-deficient (Nlrp3–/–) or caspase-1–deficient (Casp-1–/–) mice unless exogenous IL-1 is provided. Accordingly, anticancer chemotherapy turned out to be inefficient against tumors established in purinergic receptor P2rx7–/–or Nlrp3–/–or Casp1–/–hosts. Anthracycline-treated individuals with breast cancer carrying a loss-of-function allele of P2RX7 developed metastatic disease more rapidly than individuals bearing the normal allele.
    [Show full text]
  • Actions of a Series of PPADS Analogs at P2X1 and P2X3 Receptors
    DRUG DEVELOPMENT RESEARCH 53:281–291 (2001) DDR Research Article Actions of a Series of PPADS Analogs at P2X1 and P2X3 Receptors Sean G. Brown,1 Yong-Chul Kim,2 Soon-Ai Kim,2 Kenneth A. Jacobson,2 Geoffrey Burnstock,1 and Brian F. King1* 1Autonomic Neuroscience Institute, Royal Free and University College Medical School, Hampstead, London, United Kingdom 2Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland Strategy, Management and Health Policy Venture Capital Preclinical Development Clinical Development Enabling Preclinical Toxicology, Formulation Phases I-III Postmarketing Technology Research Drug Delivery, Regulatory, Quality, Phase IV Pharmacokinetics Manufacturing ABSTRACT Seven PPADS (Pyridoxal-5′-Phosphate 6-Azophenyl 2′,4′-DiSulfonate) analogs were investi- gated at Group 1 P2X receptors expressed in Xenopus oocytes. All seven analogs potently inhibited P2X1 (IC50 range, 5–32 nM) and P2X3 (IC50 range, 22–345 nM), the two Group I P2X receptor subtypes. Analogs showed greater inhibitory activity where the pyridoxal moiety of PPADS contained a 5′-phosphonate group, rather than a 5′-phosphate group. Analogs also showed greater potency where disulfonate groups were removed from, or substituted at, the azophenyl moiety. The most active analog was MRS 2257 (pyridoxal- 5′-phosphonate 6-azophenyl 3′,5′-bismethylenephosphonate) at P2X1 (IC50, 5 nM) and P2X3 (IC50, 22 nM) receptors, being 14-fold and 10-fold more potent than PPADS itself. MRS 2257 produced a nonsurmountable inhibition when tested against a range of ATP concentrations, although blockade was reversed by about 85% after 20 minutes of washout. TNP-ATP and Ip5I were equipotent with MRS 2257 at P2X1 receptors, whereas TNP-ATP was 64-fold more potent than MRS 2257 at P2X3 receptors.
    [Show full text]