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OtherCELLULAR AND MOLECULAR PHARMACOLOGY

Agonist-Independent Effect of an Allosteric Enhancer of the A1 Adenosine Receptor in CHO Cells Stably Expressing the Recombinant Human A1 Receptor

C. A. Kollias-Baker, J. Ruble, M. Jacobson, J. K. Harrison, M. Ozeck, J. C. Shryock and L. Belardinelli
Journal of Pharmacology and Experimental Therapeutics May 1997, 281 (2) 761-768;
C. A. Kollias-Baker
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J. Ruble
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M. Jacobson
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J. K. Harrison
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M. Ozeck
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J. C. Shryock
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L. Belardinelli
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Abstract

The allosteric enhancer PD 81,723, a 2-amino-3-benzoylthiophene derivative, has been shown to potentiate agonist binding to A1 adenosine receptors (A1AdoRs) and to enhance the functional effects of adenosine and adenosine analogs. The objective of this study was to determine whether the apparent agonist-independent effect of PD 81,723 observed in CHO cells stably expressing the recombinant human A1AdoR was due to the potentiation of the action of endogenous adenosine, to the presence of constitutive receptor activity and/or to the binding of PD 81,723 to the agonist binding site of the A1AdoR. The allosteric enhancer PD 81,723, the A1AdoR agonist (R)-N6-(2-phenylisopropyl)adenosine and adenosine all significantly inhibited forskolin-stimulated cAMP accumulation in intact cells and increased [35S]-5′-(γ-thio)triphosphate binding to cell membranes. The effects of adenosine on cAMP formation and [35S]-5′-(γ-thio)triphosphate binding were attenuated by adenosine deaminase, but the effects of PD 81,723 were not. In the presence of ADA, the A1AdoR antagonist 8-cyclopentyl-1,3-dipropylxanthine increased forskolin-stimulated cAMP accumulation in cells expressing the recombinant human A1AdoR but not in nontransfected CHO cells. In binding experiments, the agonist (R)-N6-(2-phenylisopropyl)adenosine, but not PD 81,723, significantly displaced the specific binding of the A1AdoR agonist [3H]-N6-cyclohexyladenosine and the antagonist [3H]-8-cyclopentyl-1,3-dipropylxanthine. The results of this study demonstrate that in CHO cells stably expressing the recombinant human A1AdoR, the agonist-independent effect of PD 81,723 is not due to potentiation of the action of endogenous adenosine or mediated by the binding of the allosteric enhancer to the agonist binding site of the recombinant human A1AdoR. It is possible that these effects are due to potentiation of constitutive receptor activity by PD 81,723.

Footnotes

  • Send reprint requests to: L. Belardinelli, M.D., University of Florida, Department of Medicine, P.O. Box 100277, Gainesville, FL 32610.

  • ↵1 This work was supported by National Institutes of Health Grant HL-50488 (L.B.) and a postdoctoral fellowship from the American Heart Association, Florida Affiliate (C.A.K.-B.).

  • ↵2 Current address: School of Veterinary Medicine, University of California, CVDLS, P.O. Box 1770, Davis, CA 95617.

  • Abbreviations:
    PD 81
    723, (2-amino-4,5-dimethyl-3-thienyl)-[3-(trifluromethyl)phenyl]methanone
    cAMP
    adenosine-3′,5′-cyclic monophosphate
    ADA
    adenosine deaminase
    GTPγS
    guanosine-5′-O-(3-thiotriphosphate)
    GDP
    guanosine-5′-diphosphate
    CPX
    8-cyclopentyl-1,3-dipropylxanthine, CPA, N6-cyclopentyl adenosine
    (R)-PIA
    (R)-N6-(2-phenylisopropyl)adenosine
    CHA
    N6-cyclohexyladenosine
    HBSS
    Hanks’ balanced salt solution
    [3H]CPX
    8-[dipropyl-2,3-3H(N)]cyclopentyl-1,3-dipropylxanthine
    [3H]CHA
    N6-[adenine-2,8-3H]cyclohexyladenosine
    [35S]GTP[S]
    [35S]-5′-(γ-thio)triphosphate
    [3H]cAMP
    [2,8-3H]adenosine-3′,5′-cyclic phosphate
    AdoR
    adenosine receptor
    G protein
    guanine nucleotide-binding protein
    CHO
    Chinese hamster ovary
    • Received December 12, 1996.
    • Accepted January 27, 1997.
  • The American Society for Pharmacology and Experimental Therapeutics
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Journal of Pharmacology and Experimental Therapeutics
Vol. 281, Issue 2
1 May 1997
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OtherCELLULAR AND MOLECULAR PHARMACOLOGY

Agonist-Independent Effect of an Allosteric Enhancer of the A1 Adenosine Receptor in CHO Cells Stably Expressing the Recombinant Human A1 Receptor

C. A. Kollias-Baker, J. Ruble, M. Jacobson, J. K. Harrison, M. Ozeck, J. C. Shryock and L. Belardinelli
Journal of Pharmacology and Experimental Therapeutics May 1, 1997, 281 (2) 761-768;

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OtherCELLULAR AND MOLECULAR PHARMACOLOGY

Agonist-Independent Effect of an Allosteric Enhancer of the A1 Adenosine Receptor in CHO Cells Stably Expressing the Recombinant Human A1 Receptor

C. A. Kollias-Baker, J. Ruble, M. Jacobson, J. K. Harrison, M. Ozeck, J. C. Shryock and L. Belardinelli
Journal of Pharmacology and Experimental Therapeutics May 1, 1997, 281 (2) 761-768;
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