Canine Mast Cell Adenosine Receptors: Cloning and Expression of the A3 Receptor and Evidence that Degranulation Is Mediated by the A2B Receptor

  1. John A. Auchampach1,
  2. Xiaowei Jin1,
  3. Tina C. Wan1,
  4. George H. Caughey2 and
  5. Joel Linden1
  1. Departments of Medicine (Cardiology), and 1Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, Virginia 22908 (J.A.A., X.J., T.C.W., J.L.), and 2Department of Medicine, Cardiovascular Research Institute, University of California at San Francisco, San Francisco, California 94143 (G.H.C.)

    Abstract

    We cloned and characterized the canine A3 adenosine receptor (AR) and examined AR-induced degranulation of the BR line of canine mastocytoma cells. Canine A3AR transcript is found predominantly in spleen, lung, liver, and testes and encodes a 314-amino acid heptahelical receptor.125I-N6-Aminobenzyladenosine binds to two affinity states of canine A3AR withKD values of 0.7 ± 0.1 and 16 ± 0.8 nm, reflecting G protein-coupled and -uncoupled receptors, respectively. Xanthine antagonists bind with similar affinities to human, canine, and rabbit receptors but with 80–400-fold lower affinities to rat A3AR. Although canine BR mastocytoma cells contain A1AR, A2BAR, and A3AR, degranulation seems to be mediated primarily by A2BARs stimulated by the nonselective agonist 5′-N-ethylcarboxamidoadenosine (NECA) but not by the A3-selective agonistN6-(3-iodobenzyl)adenosine-5′-N-methylcarboxamide. NECA-stimulated degranulation is not prevented by pertussis toxin and is blocked by enprofylline (Ki = 7 μm), an antiasthmatic xanthine with low affinity (Ki > 100 μm) for A1AR, A2AAR, and A3AR. NECA increases canine mastocytoma cell cAMP, Ca2+, and inositol trisphosphate levels; these responses are antagonized half-maximally by 7–15 μm enprofylline. The results suggest that (i) the cloned canine A3AR is structurally and pharmacologically more similar to human than to rat A3AR; (ii) the A2BAR, and not the A1AR or A3AR, is principally responsible for adenosine-mediated degranulation of canine BR mastocytoma cells; and (iii) the BR cell A2BAR couples to both Ca2+ mobilization and cAMP accumulation. Although A2B receptors play a major role in the regulation of BR mast cell degranulation, multiple AR subtypes and G proteins may influence mast cell functions.

    Footnotes

    • Send reprint requests to: Joel Linden, Ph.D., Box MR4 6012, Health Sciences Center, University of Virginia, Charlottesville, VA 22908. E-mail: jlinden{at}virginia.edu

    • ↵1 Serum-free medium was used to avoid the potential for neutralization of pertussis toxin by anti-toxin antibodies that exist within some lots of serum.

    • ↵2 X. Jin and J. Linden, unpublished observations.

    • ↵3 Mast cell mediators such as histamine can produce vasoconstriction or vasodilation. Microvascular vasoconstriction is mediated in part by histamine and thromboxane acting on vascular smooth muscle cell receptors (9). Systemic vasodilation and hypotension secondary to A3 adenosine receptor activation are mediated in part by circulating histamine, which triggers nitric oxide release from endothelial cells.

    • This work was supported by National Institutes of Health Grants RO1-HL37942 and T32-HL07284.

    • Abbreviations:
      [125I]APNEA
      N6-2-(4-amino-3-[125I]iodophenyl)adenosine
      [125I]ABA
      N6-(4-amino-3-[125I]iodobenzyl)adenosine
      [125I]AB-MECA
      N6-(4-amino-3-[125I]iodobenzyl)-adenosine-5′-N-methylcarboxamide
      IB-MECA
      N6-(3-iodobenzyl)-adenosine-5′-N-methylcarboxamide
      GTPγS
      guanosine-5′-O-(3-thio)triphosphate
      XAC
      8-(4-[(2-aminoethyl)aminocarbonylmethyloxy]-phenyl)-1,3-dipropylxanthine
      CPA
      N6-cyclopentyladenosine
      AM
      acetoxymethyl ester
      (R)-PIA
      (R)-N6-phenylisopropyladenosine
      NECA
      5′-N-ethylcarboxamidoadenosine
      I-ABOPX
      3-(4-amino-3-iodobenzyl)-8-oxyacetate-1-propyl-xanthine
      8-SPT
      8-sulfophenyltheophylline
      NBTI
      nitrobenzylthioinosine
      RT
      reverse transcription
      PCR
      polymerase chain reaction
      BSA
      bovine serum albumin
      SSC
      standard saline citrate
      SDS
      sodium dodecyl sulfate
      DMEM
      Dulbecco’s modified Eagle’s medium
      HEK
      human embryonic kidney
      InsP3
      inositol trisphosphate
      HEPES
      4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
      • Received June 17, 1997.
      • Accepted July 31, 1997.
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    1. Molecular Pharmacology November 1, 1997 vol. 52 no. 5 846-860
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