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Research ArticleArticle

Antagonist Selective Modulation of Adenosine A1 and A3 Receptor Pharmacology by the Food Dye Brilliant Black BN: Evidence for Allosteric Interactions

L. T. May, S. J. Briddon and S. J. Hill
Molecular Pharmacology April 2010, 77 (4) 678-686; DOI: https://doi.org/10.1124/mol.109.063065
L. T. May
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S. J. Briddon
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S. J. Hill
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Abstract

Allosteric binding sites on the adenosine receptor family represent potential therapeutic targets for a number of conditions involving metabolic stress. This study has identified Brilliant Black BN as a novel allosteric modulator of the adenosine A1 and A3 receptors. In addition to being a food dye and pharmaceutical excipient, Brilliant Black BN is commonly used within calcium mobilization assays to quench extracellular fluorescence. Brilliant Black BN (5–500 μM) had no significant effect on the calcium mobilization stimulated by the nonselective adenosine receptor agonist 5′-(N-ethylcarboxamido)adenosine in Chinese hamster ovary cells stably transfected with the human adenosine A1 or A3 receptor. Likewise, calcium mobilization and radioligand binding assays found that Brilliant Black BN (5–500 μM) did not significantly influence the antagonism mediated by 8-cyclopentyl-1,3-dipropylxanthine (100 nM) at the A1 receptor. In contrast, the affinity of N-[9-chloro-2-(2-furanyl)[1,2,4]-triazolo[1,5-c]quinazolin-5-yl]benzene acetamide (MRS1220) at the A3 receptor and xanthine amine congener (XAC) and XAC-X-BY630 at the A1 and A3 receptors was significantly decreased in the presence of 500 μM Brilliant Black BN. A reduction in XAC potency at the A1 and A3 receptor was achieved within 1 min of Brilliant Black BN addition, despite receptors having been pre-equilibrated with antagonist. Dissociation kinetics of the fluorescent XAC derivative, XAC-X-BY630, revealed that the decrease in affinity is probably due to a significant increase in dissociation rate of the antagonist in the presence of Brilliant Black BN. Taken together, these results suggest that Brilliant Black BN can act allosterically to modify ligand affinity at A1 and A3 receptors.

Footnotes

    fn-4
  • This work was supported by the UK Medical Research Council [Grant G0800006] and by a National Health and Medical Research Council of Australia Postdoctoral Fellowship (to L.T.M.).

  • fn-5
  • Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.

    doi:10.1124/mol.109.063065.

  • fn-6
  • ABBREVIATIONS:

    GPCR
    G protein-coupled receptor
    NECA
    5′-(N-ethylcarboxamido)adenosine
    DPCPX
    8-cyclopentyl-1,3-dipropylxanthine
    MRS1220
    N-[9-chloro-2-(2-furanyl)[1,2,4]-triazolo[1,5-c]quinazolin-5-yl]benzene acetamide
    XAC
    xanthine amine congener
    CHO
    Chinese hamster ovary
    DMEM
    Dulbecco's modified Eagle's medium
    FCS
    fetal calf serum
    AM
    acetoxymethyl ester
    HBSS
    HEPES-buffered saline solution
    PBS
    phosphate-buffered saline.

    • Received December 10, 2009.
    • Accepted January 19, 2010.
  • Copyright © 2010 The American Society for Pharmacology and Experimental Therapeutics
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Molecular Pharmacology: 77 (4)
Molecular Pharmacology
Vol. 77, Issue 4
1 Apr 2010
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Research ArticleArticle

Antagonist Selective Modulation of Adenosine A1 and A3 Receptor Pharmacology by the Food Dye Brilliant Black BN: Evidence for Allosteric Interactions

L. T. May, S. J. Briddon and S. J. Hill
Molecular Pharmacology April 1, 2010, 77 (4) 678-686; DOI: https://doi.org/10.1124/mol.109.063065

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Research ArticleArticle

Antagonist Selective Modulation of Adenosine A1 and A3 Receptor Pharmacology by the Food Dye Brilliant Black BN: Evidence for Allosteric Interactions

L. T. May, S. J. Briddon and S. J. Hill
Molecular Pharmacology April 1, 2010, 77 (4) 678-686; DOI: https://doi.org/10.1124/mol.109.063065
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