High Throughput Functional Assays for P2X Receptors

Marian T. Namovic1, Michael F. Jarvis1, Diana Donnelly‐Roberts1

1 Global Pharmaceutical Research and Development, Abbott Laboratories, Abbott Park, Illinois
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 9.15
DOI:  10.1002/0471141755.ph0915s57
Online Posting Date:  June, 2012
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Abstract

Adenosine triphosphate (ATP) activates two receptor superfamilies, metabotropic P2Y and ionotropic P2X receptors. The P2X receptors are nonselective cation channels that are widely expressed on excitable cells including neurons, glia, and smooth muscle cells. The protocols in this unit are useful for evaluating ligands that interact with P2X receptors on native cells or that are cloned and expressed in recombinant heterologous cell systems. Calcium imaging methods are described for the pharmacological characterization of fast or slowly desensitizing P2X receptors. While these methods are readily applicable to a wide variety of ligand‐gated ion channels, the protocols provided herein detail how they can be used to measure activation of homomeric P2X3 (fast desensitizing) and heteromeric P2X2/3 (slowly desensitizing) receptors. Appropriate agonists and/or calcium dyes can be substituted to assess activity at other P2X receptor subtypes. An additional protocol is provided for measuring P2X7 receptor‐mediated pore formation in THP‐1, a native human acute monocytic leukemia cell line that can be used to study homomeric P2X7 (non‐desensitizing) receptors that are expressed on macrophages and microglial cells. Curr. Protoc. Pharmacol. 57:9.15.1‐9.15.21. © 2012 by John Wiley & Sons, Inc.

Keywords: P2X1; P2X2; P2X2/3; P2X3; P2X4; P2X7; FLIPR; YO‐PRO

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Measurement of Calcium Dynamics of Slow‐Desensitizing P2 Receptors—P2X2/3, P2X2, P2X4—and Non‐Desensitizing P2 Receptor P2X7 using FLIPR384 Fluorometric Imaging
  • Alternate Protocol 1: Measurement of Calcium Dynamics in Rapid‐Desensitizing Homomeric P2 Receptors: P2X3, P2X1
  • Basic Protocol 2: Measurement of P2X7‐Induced Pore Formation in a FLIPR384
  • Alternate Protocol 2: Measurement of BzATP‐Mediated Pore Formation in Native Human P2X7‐Expressing THP‐1 Cells in a FLIPR384
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Measurement of Calcium Dynamics of Slow‐Desensitizing P2 Receptors—P2X2/3, P2X2, P2X4—and Non‐Desensitizing P2 Receptor P2X7 using FLIPR384 Fluorometric Imaging

  Materials
  • 1321N1 human astrocytoma cells stably expressing recombinant P2 receptors with use of various poly(A)+ RNAs supplied by Clontech (Bianchi et al., ; Lynch et al., )
  • Complete growth medium for 1321N1 cells expressing slow/non‐desensitizing P2X receptors (see recipe; same recipe for both slow and non‐desensitizing P2X receptors)
  • Dulbecco's phosphate‐buffered saline (DPBS) with Ca+2, Mg+2 and 1 mg/ml D‐glucose (Invitrogen, cat. no. 14287‐072)
  • 0.25% (1×) trypsin with EDTA (Invitrogen, cat. no. 25200‐056)
  • Trypan blue stain (Invitrogen, cat. no. 15250‐061)
  • Test compounds: agonists and antagonists, e.g.:
    • ATP (adenosine 5′‐triphosphate disodium salt hydrate; Sigma, cat. no. A‐2383); agonist
    • BzATP [2′(3′)‐O‐(4‐benzoylbenzoyl)adenosine 5′‐triphosphate triethylammonium salt; Sigma, cat. no. B6396); agonist
    • α,β‐methyleneadenosine 5′‐triphosphate lithium salt; AMP‐CPP; Sigma, cat. no. M‐6517); agonist
    • UTP [uridine 5′‐(trihydrogen diphosphate) sodium salt from Saccharomyces cerevisiae; Sigma, cat. no. U‐4125]; agonist
    • Diadenosine polyphosphate (Ap nA, where n = 3 to 6; Sigma; also see Bianchi et al., ); agonists
    • PPADS (pyridoxal phosphate‐6‐azophenyl‐2‐4‐disulfonic acid; Sigma, cat. no. P178); antagonist
    • KN‐62 (1‐[N,O‐bis(5‐isoquinolinesulfonyl)‐N‐methyl‐L‐tyrosyl]‐4‐phenylpiperazine; Sigma, cat. no. I2142); antagonist
    • BBG (Brilliant Blue G; Sigma, cat. no. B0770); antagonist
    • PPNDS (pyridoxal‐5′‐phosphate‐6‐(2′‐naphthylazo‐6′‐nitro‐4′,8′‐disulfonate) tetrasodium salt; Tocris, cat. no. 1309); antagonist
    • MRS 2159 (Sigma, cat. no. M7684); antagonist
    • Reactive Blue 2 (Sigma, cat. no. R115); antagonist
    • NF 279 (Tocris, cat. no. 1199); antagonist
    • A 438079 hydrochloride (Tocris, cat. no. 2972); antagonist
    • A 740003 (Tocris, cat. no. 7301); antagonist
  • FLUO‐4 AM (Invitrogen, cat. no. F14202)
  • Dimethylsulfoxide (DMSO; Sigma)
  • 162‐cm2 (T‐162) cell culture flasks, canted with vented gas‐permeable caps, sterile and nonpyrogenic, polystyrene (Costar)
  • 50‐ml conical centrifuge tubes (e.g., BD Falcon)
  • Hemacytometer INCYTO C‐Chip Disposable (VWR, cat. no. DHCN012)
  • Centrifuge, and microtiter plate adapter
  • 96‐well (cat. no. 35‐4640) or 384‐well (cat. no. 35‐4396) black‐walled, clear‐bottom poly‐D‐lysine coated plates (Becton Dickinson)
  • 96‐well (cat. no. 3897) or 384‐well (cat. no. 3680) V‐bottom plates (Corning)
  • FLIPR384 fluorometric imaging plate reader (Black Dog Technical Services, http://www.blackdogts.com/; also see unit 9.2)
  • Black pipet tips for FLIPR (Molecular Devices)
  • Automated cell plate washer (e.g., Aquamax 2000, Molecular Devices)
  • Analysis/spreadsheet software, (e.g., GraphPad Prism, Microsoft Excel)
  • Additional reagents and equipment for FLIPR (unit 9.2)

Alternate Protocol 1: Measurement of Calcium Dynamics in Rapid‐Desensitizing Homomeric P2 Receptors: P2X3, P2X1

  • Complete growth medium for 1321N1 cells expressing rapid desensitizing P2X receptors (see recipe)

Basic Protocol 2: Measurement of P2X7‐Induced Pore Formation in a FLIPR384

  Materials
  • 1321N1 human astrocytoma cells stably expressing recombinant P2X7 receptors with use of various poly(A)+ RNAs supplied by Clontech (Bianchi et al., ; Lynch et al., )
  • Complete growth medium for 1321N1 cells expressing slow desensitizing P2X receptors (see recipe)
  • 0.25% (1×) trypsin with EDTA (Invitrogen, cat. no. 25200‐056)
  • Trypan blue stain (Invitrogen, cat. no. 15250‐061)
  • Dulbecco's phosphate‐buffered saline, DPBS, without Ca2+ and Mg2+ (Invitrogen, cat. no. 14190), supplemented with 1 mg/ml D‐(+)‐glucose
  • YO‐PRO‐1 iodide (491/509; Invitrogen, cat. no. Y3603)
  • Test compounds: agonists and antagonists, e.g.:
    • BzATP [2′(3′)‐O‐(4‐benzoylbenzoyl)adenosine 5′‐triphosphate triethylammonium salt; Sigma, cat. no. B6396); agonist
    • PPADS (pyridoxal phosphate‐6‐azophenyl‐2‐4‐disulfonic acid; Sigma, cat. no. P178); antagonist
    • KN‐62 (1‐[N,O‐bis(5‐isoquinolinesulfonyl)‐N‐methyl‐L‐tyrosyl]‐4‐phenylpiperazine; Sigma, cat. no. I2142); antagonist
    • BBG (Brilliant Blue G; Sigma, cat. no. B0770); antagonist
    • PPNDS (pyridoxal‐5′‐phosphate‐6‐(2′‐naphthylazo‐6′‐nitro‐4′,8′‐disulfonate) tetrasodium salt; Tocris, cat. no. 1309); antagonist
    • MRS 2159 (Sigma, cat. no. M7684); antagonist
    • Reactive Blue 2 (Sigma, cat. no. R115); antagonist
    • NF 279 (Tocris, cat. no. 1199); antagonist
    • A 438079 hydrochloride (Tocris, cat. no. 2972); antagonist
    • A 740003 (Tocris, cat. no. 7301); antagonist
  • Dimethylsulfoxide (DMSO; Sigma)
  • 162‐cm2 (T‐162) cell culture flasks, canted with vented gas‐permeable caps, sterile and nonpyrogenic, polystyrene (Costar)
  • 50‐ml conical centrifuge tubes (e.g., BD Falcon)
  • Hemacytometer INCYTO C‐Chip Disposable (VWR, cat. no. DHCN012)
  • Centrifuge, and microtiter plate adapter
  • 96‐well (cat. no. 35‐4640) or 384‐well (cat. no. 35‐4396) black‐walled, clear‐bottom poly‐D‐lysine coated plates (Becton Dickinson)
  • 96‐well (cat. no. 3897) or 384‐well (cat. no. 3680) V‐bottom plates (Corning)
  • FLIPR384 fluorometric imaging plate reader (Black Dog Technical Services, http://www.blackdogts.com/; also see unit 9.2)
  • Black pipet tips for FLIPR (Molecular Devices)
  • Automated cell plate washer (e.g., Aquamax 2000, Molecular Devices)
  • Analysis/spreadsheet software, (e.g., GraphPad Prism, Microsoft Excel)
  • Additional reagents and equipment for FLIPR (unit 9.2)

Alternate Protocol 2: Measurement of BzATP‐Mediated Pore Formation in Native Human P2X7‐Expressing THP‐1 Cells in a FLIPR384

  • THP‐1 cells (ATCC cat. no. TIB‐202)
  • Complete growth medium for THP‐1 monocytic cells (see recipe)
  • Lipopolysaccharide (LPS) from E. coli Strain 0111:B4 (Sigma, cat. no. L‐5293‐2ml)
  • Human interferon gamma (IFN‐γ; R&D Systems, cat. no. AB‐285‐NA)
  • YO‐PRO‐1 iodide (491/509; Invitrogen, cat. no. Y3603)
  • Dulbecco's phosphate‐buffered saline (DPBS) without Ca2+ and Mg2+ (Invitrogen, cat. no. 14190), supplemented with 1 mg/ml D‐(+)‐glucose
  • Cell dissociation buffer (Invitrogen, cat. no. 13150)
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Figures

Videos

Literature Cited

   Bianchi, B.R., Lynch, K.J., Touma, E., Niforatos, W., Burgard, E.C., Alexander, K.M., Park, H.S., Yu, H., Metzger, R., Kowaluk, E.A., Jarvis, M.F., and van Biesen, T. 1999. Pharmacological characterization of recombinant human and rat P2X receptor subtypes. Eur. J. Pharmacol. 376:127‐138.
   Burnstock, G. 2007. Physiology and pathophysiology of purinergic neurotransmission. Physiol. Rev. 87:659‐797.
   Burnstock, G., Krugel, U., Abbracchio, M.P., and Illes, P. 2011. Purinergic signaling: From normal behavior to pathological brain function. Prog. Neurobiol. 95:229‐274.
   Donnelly‐Roberts, D.L. and Jarvis, M.F. 2007. Discovery of P2X7 receptor‐selective antagonists offers new insights into P2X7 receptor function and indicates a role in chronic pain states. Br. J. Pharmacol. 151:571‐579.
   Donnelly‐Roberts, D.L., Namovic, M., Faltynek, C.R., and Jarvis, M.F. 2004. Mitogen‐activated protein kinase and caspase signaling pathways are required for P2X7 receptor (P2X7R)‐induced pore formation in Human THP‐1 cells. J. Pharmacol. Exp. Ther. 308:1053‐1061.
   Donnelly‐Roberts, D.L., Namovic, M., Han, P., and Jarvis, M.F. 2009. Mammalian P2X7 receptor pharmacology: A comparison of recombinant mouse, rat and human P2X7 receptors. Br. J. Pharmacol. 157:1203‐1214.
   Faria, R.X., DeFarias, F.P., and Alves, L.A. 2005. Are second messengers crucial for opening the pore associated with P2X7 receptor? Am. J. Physiol. Cell. Physiol. 288:C260‐C271.
   Gever, J.R., Cockayne, D.A., Dillon, M.P., Burnstock, G., and Ford, A.P. 2006. Pharmacology of P2X channels. Pflugers Arch. 452:513‐537.
   Honore, P., Donnelly‐Roberts, D., Namovic, M.T., Hsieh, G., Zhu, C.Z., Mikusa, J.P., Hernandez, G., Zhong, C., Gauvin, D.M., Chandran, P., Harris, R., Medrano, A.P., Carroll, W., Marsh, K., Sullivan, J.P., Faltynek, C.R., and Jarvis, M.F. 2006. A‐740003 (N‐(1‐cyanoimino) (5‐quinolinylamino) methyl]amino‐2,2 dimethylpropyl)‐2‐(3,4‐dimethoxyphenyl) acetamide, a novel and selective P2X7 receptor antagonist dose‐dependently reduces neuropathic pain in the rat. J. Pharmacol. Exp. Ther. 319:1376‐1385.
   Humphreys, B.D. and Dubyak, G.R. 1996. Induction of the P2z/P2X7 nucleotide receptor and associated phospholipase D activity by lipopolysaccharide and IFN‐γ in the human THP‐1 monocytic cell line. J. Immunol. 157:5627‐5637.
   Jacobson, K.A., Jarvis, M.F., and Williams, M. 2002. Perspective: Purine and pyrimidine (P2) receptors as drug targets. J. Med. Chem. 45:4057‐4093.
   Jarvis, M.F. 2010. The neural‐glial purinergic receptor ensemble in chronic pain states. Trends Neurosci. 33:48‐57.
   Jarvis, M.F. and Khakh, B.S. 2009. ATP‐gated P2X cation‐channels. Neuropharmacology 56:208‐215.
   Khakh, B.S. and North, R.A. 2006. P2X receptors as cell‐surface ATP sensors in health and disease. Nature 442:527‐532.
   Lynch, K.L., Touma, E., Niforatos, W., Kage, K.L., Burgard, E.C., van Biesen, T., Kowaluk, E.A., and Jarvis, M.F. 1999. Molecular and functional characterization of human P2X(2) receptors. Mol. Pharmacol. 56:1171‐1181.
   Neelands, T.R., Burgard, E.C., Uchic, M.E., McDonald, H.A., Niforatos, W., Faltynek, C.R., Lynch, K.J., and Jarvis, M.F. 2003. 2′, 3′‐O‐(2,4,6,trinitrophenyl)‐ATP and A‐317491 are competitive antagonists at a slowly desensitizing chimeric human P2X3 receptor. Br. J. Pharmacol. 140:202‐210.
   Nelson, D.W., Gregg, R.J., Kort, M.E., Perez‐Medrano, A., Voight, E.A., Wang, Y., Grayson, G., Namovic, M.T., Donnelly‐Roberts, D.L., Niforatos, W., Honore, P., Jarvis, M.F., Faltynek, C.R., and Carroll, W.A. 2006. Structure‐activity relationship studies on a series of novel, substituted 1‐benzyl‐5‐phenyltetrazole P2X7 antagonists. J. Med. Chem. 49:3659‐3666.
   North, A.R. 2002. Molecular physiology of P2X receptors. Physiol. Rev. 82:1013‐1067.
   Orriss, I.R., Knight, G.E., Utting, J.C., Taylor, S.E., Burnstock, G., and Arnett, T.R. 2009. Hypoxia stimulates vesicular ATP release from rat osteoblasts. J. Cell Physiol. 220:155‐162.
   Pelegrin, P. and Surprenant, A. 2006. Pannexin‐1 mediates large pore formation and interleukin‐1b release by the ATP‐gated P2X7 receptor. EMBO J. 25:5071‐5082.
   Suprenant, A., Rassendren, F., Kawashima, E., North, R.A., and Buell, G. 1996. The Cytolytic P2z receptor for extracellular ATP identified as a P2X receptor (P2X7). Science 272:735‐738.
Internet Resources
   http://assay.nih.gov/assay/index.php/Table_of_Contents#Flipr.E2.84.A2_Assays_to_Measure_GPCR_and_Ion_Channel_Targets
  Web site describing FLIPR assays to measure GPCR and ion channels, giving information on FLIPR usage.
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