Characterization of P1 (Adenosine) Purinoceptors

互联网2013-12-31

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Abstract
Table of Contents
Materials
Figures
Literature Cited

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 , A_2A , and A₃ receptors. Pharmacological characterization of the low?affinity A_2B 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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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 A₁ receptor expressed in Sf 9 cells (e.g., Research Biochemicals or Biosignal)
A₁ 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
[³ H]Cyclohexyladenosine ([³ H]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)

CO₂ 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

	A₁ [³ H]CHA	A_2A [³ H]CGS21680	A_2B [³ H] PSB‐603	A₃ [¹²⁵ I]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

^a Saturation binding parameters (K _d and B_max ) for A₁ and A_2A are for rat brain. The A_2B and A₃ binding parameters (K _d , B_max , and K _i ) are for the recombinant human receptors.

^b K _i (nM) values for A₁ and A_2A receptors from Jarvis et al. ( ) and Jacobson et al. ( ), for A_2B receptors from Borrmann et al. ( ), and for A₃ receptors from Patel et al. ( ).

^c Abbreviations: 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, N ⁶ ‐cyclohexyladenosine; 2‐CADO, 2‐chloroadenosine; CPA, N ⁶ ‐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, N ⁶ ‐(R )‐phenylisopropyladenosine; S‐PIA, N ⁶ ‐(S )‐phenylisopropyladeno‐sine; XAC, xanthine amino acid congener.

^d ZM 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 MgCl₂ , 4°C
Adenosine deaminase (Type III; Boehringer Mannheim)
Acetone/dry ice bath
[³ H]N⁶ ‐Cyclohexyladenosine ([³ H]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)

CO₂ 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 A₃ receptor expressed in HEK‐293 cells (e.g., Research Biochemicals or Receptor Biology)
A₃ assay buffer: 50 mM Tris·Cl (pH 8.25 at 4°C; appendix 2A )/10 mM MgCl₂ /1 mM EDTA containing 2 IU/ml adenosine deaminase (type III; Boehringer Mannheim), 4°C
[¹²⁵ I]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)

CO₂ 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

Figure 1.9.1 (A ) Representative autoradiographic image of specific [³ H]CHA (1 nM) binding to rat brain sagittal sections (Jarvis, ). (B ) Representative autoradiographic image of specific [³ H]CGS 21680 (5 nM) binding to ADO A_2A receptors in an adjacent rat brain sagittal section. Specific binding is revealed by digital subtraction autoradiography, in which the image of specific binding is obtained through subtraction of the linearized nonspecific binding image from the total binding image. Dark areas represent regions of densely bound radioligand, whereas lighter areas indicate little or no specific binding. Abbreviations: C, cerebral cortex; CP, caudate‐putamen; NA, nucleus acumbens; OT, olfactory tubercle; TH, thalamus; CA1 and CA3, CA1 and CA3 regions of hippocampus; CG, cerebellum, granular layer; CM, cerebellum, molecular layer.

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Videos

Literature Cited

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	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.
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	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.
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	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.
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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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Characterization of P1 (Adenosine) Purinoceptors

Abstract

Table of Contents

Materials

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

Figures

Videos

Literature Cited