Characterization of P1 (Adenosine) Purinoceptors

Michael F. Jarvis1

1 AbbVie, Global Research and Development, North Chicago, Illinois
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 1.9
DOI:  10.1002/0471141755.ph0109s62
Online Posting Date:  October, 2013
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Abstract

The purine nucleoside adenosine (ADO) is an important modulator of cellular function in mammalian tissues, modulating cellular function and neuronal excitability via interactions with different cell surface receptor subtypes that are heterogeneously distributed in both the mammalian CNS and peripheral tissues. Four ADO receptor subtypes have been cloned and characterized. Described in this unit are three radioligand binding assays for pharmacological characterization of the high‐affinity ADO receptor subtypes A1, A2A, and A3 receptors. Pharmacological characterization of the low‐affinity A2B receptor has been enabled by the use of tritiated xanthine PSB‐603. Because receptor localization is an important criterion for differentiation of receptor subtypes, a support protocol that describes the methodology for the localization of ADO receptors in rat brain tissue using autoradiography is also included. Curr. Protoc. Pharmacol. 62:1.9.1‐1.9.16. © 2013 by John Wiley & Sons, Inc.

Keywords: adenosine; G protein–coupled receptor; neuromodulation

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Determination of [3H]CHA Binding to A1 Receptors
  • Basic Protocol 2: Determination of [3H]CGS 21680 Binding to A2A Receptors
  • Basic Protocol 3: Determination of [125I]AB‐MECA Binding to A3 Receptors
  • Support Protocol 1: Autoradiographic Localization of ADO Receptor Subtypes
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Determination of [3H]CHA Binding to A1 Receptors

  Materials
  • 200‐ to 250‐g adult male Sprague‐Dawley (TAC:SD) rats or recombinant rat A 1 receptor expressed in Sf9 cells (e.g., Research Biochemicals or Biosignal)
  • A 1 assay buffer: 50 mM Tris·Cl, pH 7.4 at 23°C ( appendix 2A), 4°C
  • Adenosine deaminase (type III; Boehringer Mannheim)
  • Acetone/dry ice bath
  • [3H]Cyclohexyladenosine ([3H]CHA; 20 to 30 Ci/mmol; NEN Life Sciences)
  • Receptor‐selective ADO ligand (see Tables 1.9.1 and 1.9.2) for use as reference compound
  • 2‐chloroadenosine (2‐CADO; Research Biochemicals)
  • Scintillation cocktail, e.g., Aquasol 2 (NEN Life Sciences)
  • CO 2 source
  • Polytron homogenizer (Brinkmann), or equivalent
  • High‐speed centrifuge (e.g., Sorvall R2‐5B, Du Pont), 4°C
  • 12 × 75–mm polypropylene test tubes or 96‐well microtiter plates
  • M‐48 cell harvester (Brandel), or equivalent, or Harvester 96 (Tomtec), or equivalent
  • Whatman GF/B glass‐fiber filters
  • Liquid scintillation counter (e.g., Beckman) and 7‐ml scintillation vials or microtiter plate–based scintillation counter (e.g., Beta‐plate and/or MicroBeta scintillation counter; LKB, Wallac)
  • Nonlinear regression curve‐fitting program, e.g., RS/1 (Bolt, Beranek, and Newman) or Prism (GraphPad Software)
  • Additional reagents and equipment for carrying out protein concentration assay ( appendix 3A)
Table 1.9.2   MaterialsComparison of the Activity of Various Adenosine Agonists and Antagonists in Inhibiting Radioligand Binding to High‐Affinity Adenosine Receptor Subtypes

A 1 [3H]CHA A 2A [3H]CGS21680 A 2B [3H] PSB‐603 A 3 [125I]AB‐MECA
K d (nM) 1 15 0.4 1
B max (fmol/mg protein) 350 370 500 50
Compound K i (nM)
Agonists
CHA 2 685 ND 1500
CPA 1 890 ND 790
NECA 6 12 2 100
CGS21680 2600 14 >10,000 1500
2‐CADO 14 120 21 ND
R‐PIA 2 410 ND 200
S‐PIA 29 3020 ND ND
AP‐NECA 14 172 ND 25
IB‐MECA 54 56 ND 10
Antagonists
DPCPX 0.5 260 ND 200
XAC 4 50 ND >10,000
CPT 11 1050 ND ND
CGS15943 22 2 0.03 8
ZM241385 755 1 ND >100,000

 aSaturation binding parameters (K d and B max) for A 1 and A 2A are for rat brain. The A 2B and A 3 binding parameters (K d, B max, and K i) are for the recombinant human receptors.
 bK i (nM) values for A 1 and A 2A receptors from Jarvis et al. ( ) and Jacobson et al. ( ), for A 2B receptors from Borrmann et al. ( ), and for A 3 receptors from Patel et al. ( ).
 cAbbreviations: APNEA, N‐[2‐(4‐aminophenyl)ethyl]adenosine; CGS 21680, (2‐[p‐(2‐carboxyethyl)phenethylamino]‐5′‐N‐ethylcarboxamidoadenosine; CGS 15943, 9‐chloro‐2‐(2‐furyl)‐[1,2,4]‐triazolo[1,5‐c]quinazolin‐5‐amine; CHA, N6‐cyclohexyladenosine; 2‐CADO, 2‐chloroadenosine; CPA, N6‐cyclopentyladenosine; CPT, 8‐cyclopentyltheo‐phylline; DPCPX, 1,3‐dipropyl‐8‐cyclopentylxanthine; IB‐MECA, iodobenzylmethylethylcarboxyamidoadenosine; ND, not determined; NECA, 5′‐N‐ethylcarboxamidoadenosine; PSB‐603, 8‐(4‐[4‐{4‐chlorophenyl}piperazine‐1‐sulfonyl]phenyl)‐1‐propylxanthine; R‐PIA, N6‐(R)‐phenylisopropyladenosine; S‐PIA, N6‐(S)‐phenylisopropyladeno‐sine; XAC, xanthine amino acid congener.
 dZM 241385 is available from Tocris Cookson; all other compounds listed are available from RBI (see ).

Basic Protocol 2: Determination of [3H]CGS 21680 Binding to A2A Receptors

  Materials
  • 200‐ to 250‐g adult male Sprague‐Dawley (TAC:SD) rats or recombinant human A 2A receptor expressed in HEK‐293 cells (e.g., Research Biochemicals or Biosignal)
  • 50 mM Tris·Cl, pH 7.4 at 23°C ( appendix 2A), 4°C
  • A 2A assay buffer: 50 mM Tris·Cl (pH 7.4 at 23°C)/10 mM MgCl 2, 4°C
  • Adenosine deaminase (Type III; Boehringer Mannheim)
  • Acetone/dry ice bath
  • [3H]N6‐Cyclohexyladenosine ([3H]CGS 21680; 20 to 30 Ci/mmol; NEN Life Sciences)
  • Receptor‐selective ADO ligand for use as reference compound (see Tables 1.9.1 and 1.9.2; available from Research Biochemicals)
  • 2‐chloroadenosine (2‐CADO; Research Biochemicals)
  • Scintillation cocktail, e.g., Aquasol 2 (NEN Research Products)
  • CO 2 source
  • Polytron homogenizer (Brinkmann)
  • High‐speed centrifuge (e.g., Sorvall RC‐5B, NEN Life Sciences), 4°C
  • 12 × 75–mm polypropylene test tubes or 96‐well microtiter plates
  • MK‐48 cell harvester (Brandel), or equivalent, or Harvester 96 (Tomtec), or equivalent
  • Whatman GF/B glass‐fiber filters
  • Liquid scintillation counter (e.g., Beckman) and 7‐ml scintillation vials or microtiter plate–based scintillation counter (e.g., Beta‐plate and/or MicroBeta scintillation counter; LKB, Wallac)
  • Nonlinear regression curve‐fitting program, e.g., RS/1 (Bolt, Beranek, and Newman) or Prism (GraphPad Software)
  • Additional reagents and equipment for protein concentration assay ( appendix 3A)

Basic Protocol 3: Determination of [125I]AB‐MECA Binding to A3 Receptors

  Materials
  • Recombinant human A 3 receptor expressed in HEK‐293 cells (e.g., Research Biochemicals or Receptor Biology)
  • A 3 assay buffer: 50 mM Tris·Cl (pH 8.25 at 4°C; appendix 2A)/10 mM MgCl 2/1 mM EDTA containing 2 IU/ml adenosine deaminase (type III; Boehringer Mannheim), 4°C
  • [125I]AB‐MECA (2200 Ci/mmol; Amersham)
  • Receptor‐selective ADO ligand for use as reference compound (see Tables 1.9.1 and 1.9.2; available from Research Biochemicals)
  • (R)‐Phenylisopropyladenosine (R‐PIA; RBI or Sigma)
  • 12 × 75–mm polypropylene test tubes
  • M‐48 cell harvester (Brandel), or equivalent, or Harvester 96 (Tomtec), or equivalent
  • Whatman GF/B glass‐fiber filters
  • Gamma detector (e.g., Cobra Auto‐Gamma, Packard) or microtiter plate–based scintillation counter (e.g., Beta‐plate and/or MicroBeta scintillation counter; LKB, Wallac)
  • Nonlinear regression curve‐fitting program: e.g., RS/1 (Bolt, Beranek, and Newman) or Prism (GraphPad Software)
  • Additional reagents and equipment for protein concentration assay ( appendix 3A)

Support Protocol 1: Autoradiographic Localization of ADO Receptor Subtypes

  Materials
  • 200‐ to 250‐g adult male Sprague‐Dawley rats (TAC:SD)
  • PBS/sucrose (see recipe)
  • Isopentane, −10°C
  • Gelatin‐subbed slides (see recipe)
  • Assay buffer: 50 mM Tris·Cl buffer, pH 7.4 at 23°C ( appendix 2A), 37°C, 23°C, and 4°C
  • Adenosine deaminase (Type III; Boehringer Mannheim)
  • Kodak D‐19 developer (Eastman Kodak)
  • Kodak fixer (Eastman Kodak)
  • CO 2 source
  • Model OTF/AS/MR/EC cryostat (Bright/Hacker)
  • Microscope slide mailing tubes (Lab‐Tek), optional
  • Tritium‐sensitive film (Bromma)
  • RAS‐1000 video‐based densitometer (Amersham), or equivalent
  • Radioactive microscales (Amersham; optional)
  • Additional reagents and equipment for carrying out binding assays for labeling adenosine receptors (Basic Protocols protocol 11, protocol 22, and protocol 33)
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Figures

Videos

Literature Cited

  Borrmann, T., Hinz, S., Bertarelli, D.G.G., Li, W., Florin, N.C., Scheiff, A.B., and Müller, C.E. 2009. 1‐Alkyl‐8‐(piperazine‐1‐sulfonyl)phenylxanthines: Development and characterization of adenosine A2B receptor antagonists and a new radioligand with subnanomolar affinity and subtype specificity. J. Med. Chem. 52:3994‐4006.
  Bradford, M.M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein‐dye binding. Anal. Biochem. 72:248‐254.
  Bruns, R.F., Lu, G.H., and Pugsley, T.A. 1986. Characterization of the A2 adenosine receptor labeled by [3H]NECA in rat striatal membranes. Mol. Pharmacol. 29:331‐346.
  Burnstock, G. and Verkhratsky, A. 2012. Purinergic signaling. WIREs Membr. Transp. Signal. 1:116‐125.
  Cunha, R.A., Johansson, B., Constantino, M.D., Sebastiao, A.M., and Fredholm, B.B. 1996. Evidence for high affinity binding sites for the adenosine agonist [3H]CGS 21680 in rat hippocampus and cerebral cortex that are different from striatal A2A receptors. Naunyn Schmiedebergs Arch. Pharmacol. 349:374‐380.
  Dulla, C.G. and Masino, S.A. 2013. Physiologic and metabolic regulation of adenosine: Mechanisms. In Adenosine. A Key Link between Metabolism and Brain Activity. (S. Masino and D. Boison, eds.) pp. 87‐107. Springer, New York.
  Eckle, T., Krahn, T., Grenz, A., Köhler, D., Mittelbronn, M., Ledent, C., Jacobson, M.A., Osswald, H., Thompson, L.F., Unertl, K., and Eltzschig, H.K. 2007. Cardioprotection by ecto‐5′‐nucleotidase (CD73) and A2B adenosine receptors. Circulation 115:1581‐1590.
  Emson, P.C. and Koob, G. 1978. The origin and distribution of dopamine‐containing afferents to the rat frontal cortex. Brain Res. 142:249‐267.
  Fredholm, B.B., IJzerman, A.P., Jacobson, K.A., Linden, J., and Müller, C.E. 2011. International Union of Basic and Clinical Pharmacology. LXXXI. Nomenclature and classification of adenosine receptors—An update. Pharmacol. Rev. 63:1‐34.
  Gessi, S., Merighi, S., Varani, K., and Borea, P.A. 2011. Adenosine receptors in health and disease. Adv. Pharmacol. 61:41‐75.
  Göblyös, A. and Ijzerman, A.P. 2011. Allosteric modulation of adenosine receptors. Biochim. Biophys. Acta 1808:1309‐1318.
  Hutchison, K.A., Nevins, B., Perini, F., and Fox, I.H. 1990. Soluble and membrane‐associated low‐affinity adenosine binding proteins (adenotin): Properties and homology with mammalian and avian stress proteins. Biochemistry 29:5138‐5144.
  Jacobson, K., van Galen, P., and Williams, M. 1992. Adenosine receptors: Pharmacology, structure‐activity relationships and therapeutic potential. J. Med. Chem. 35:407‐422.
  Jacobson, K.A., Kim, H.O., Siddiqi, S.M., Olah, M.E., Stiles, G.I., and von Lubitz, D.K.J.E. 1995. A3‐adenosine receptors: Design of selective ligands and therapeutic prospects. Drugs Future 20:689‐699.
  Jacobson, K.A., Gao, Z.‐G., Göblyös, A., and Ijzerman, A.P. 2011. Allosteric modulation of purine and pyrimidine receptors. Adv. Pharmacol. 61:187‐220.
  Jarvis, M.F. 1988. Autoradiographic localization and characterization of brain adenosine receptor subtypes. In Receptor Localization: Ligand Autoradiography (F. Leslie and C.A. Altar, eds.) pp. 95‐113. Alan R. Liss, New York.
  Jarvis, M.F. 1997. Psychomotor aspects of adenosine receptor activation. In Purinergic Approaches in Experimental Therapeutics (K.A. Jacobson and M.F. Jarvis eds.) pp. 405‐421. Wiley‐Liss, New York.
  Jarvis, M.F. and Saltzman, A. 1993. [3H]5′‐N‐ethylcarboxamidoadenosine selectively labels the low‐affinity adenosine‐binding protein, adenotin, on intact Chinese hamster ovary cells. Drug Dev. Res. 29:305‐309.
  Jarvis, M.F., Schulz, R., Hutchinson, A., Do, U., Sills, M., and Williams, M. 1989. [3H]CGS 21680, a selective A2 adenosine receptor agonist directly labels A2 receptors in rat brain. J. Pharmacol. Exp. Ther. 251:888‐893.
  Kim, Y.C., Ji, X.D., and Jacobson, K.A. 1996. Derivatives of the triazoloquinazoline adenosine antagonist (CGS 15943) are selective for the human A3 receptor subtype. J. Med. Chem. 39:4142‐4148.
  Müller, C.E. and Jacobson, K.A. 2011. Recent developments in adenosine receptor ligands. and their potential as novel drugs. Biochim. Biophys. Acta 1808:1290‐1308.
  Patel, M., Harris, C., and Lundstrom, K. 1997. The binding of [125I]AB‐MECA to the human cloned adenosine A3 receptor using the Semliki Forest Virus Expression system. Drug Dev. Res. 40:35‐40.
  Paxinos, G. and Watson, C. 1986. The Rat Brain in Stereotaxic Coordinates. Academic Press, New York.
  Sherman, L.P. and Weaver, D.R. 1997. [125]4‐Aminobenzyl‐5′‐N‐methylcarboxamidoadeno‐sine ([125I]AB‐MECA) labels multiple adenosine receptor subtypes in rat brain. Brain Res. 745:10‐20.
  Williams, M. 1995. Purinoceptors in Central Nervous System Function: Targets for Therapeutic Intervention. In Psychopharmacology: The Fourth Generation of Progress (F.E. Bloom and D.J. Kupher, eds.) pp. 643‐655. Raven Press, New York.
  Williams, M. and Jarvis, M.F. 1989. Biochemical approaches to drug discovery and characterization. In Modern Drug Discovery Technologies (C.R. Clarke and W.H. Moos, eds.) pp. 129‐166. VCH Publishers, New York.
  Williams, M. and Risley, E.A. 1980. Biochemical characterization of putative purinergic receptors by using 2‐chloro‐[3H]adenosine, a stable analog of adenosine. Proc. Natl. Acad. Sci. U.S.A. 77:6892‐6896.
Key References
  Fredholm et al., 2011. See above.
  Müller and Jacobson, 2011 See above.
Internet Resources
  http://mgddk1.niddk.nih.gov/
  Web site of the NIH Molecular Recognition Section.
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