title: Canine mast cell adenosine receptors: cloning and expression of the A3 receptor and evidence that degranulation is mediated by the A2B receptor.
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 with KD 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 agonist N6-(3-iodobenzyl)adenosine-5'-N-methylcarboxamide. NECA-stimulated degranulation is not prevented by pertussis toxin and is blocked by enprofylline (Ki = 7 microM), an antiasthmatic xanthine with low affinity (Ki > 100 microM) 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 microM 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.
authors.affiliation: Departments of Medicine (Cardiology), University of Virginia, Charlottesville, Virginia 22908, USA.
authors.forename: J A
authors.initials: JA
authors.lastname: Auchampach
authors.forename: X
authors.initials: X
authors.lastname: Jin
authors.forename: T C
authors.initials: TC
authors.lastname: Wan
authors.forename: G H
authors.initials: GH
authors.lastname: Caughey
authors.forename: J
authors.initials: J
authors.lastname: Linden
mesh_terms: D000225:Adenine / Q000031:analogs & derivatives / Q000494:pharmacology; D000241:Adenosine / Q000031:analogs & derivatives / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002118:Calcium / Q000378:metabolism; D002522:Chlorocebus aethiops; D018076:DNA, Complementary / Q000235:genetics* / Q000378:metabolism; D015226:Dinucleoside Phosphates / Q000494:pharmacology; D004285:Dogs; D008407:Mast Cells / Q000737:chemistry* / Q000502:physiology; D012515:Mast-Cell Sarcoma / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000235:genetics* / Q000378:metabolism; D009636:Norbornanes / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D043684:Receptor, Adenosine A3; D018047:Receptors, Purinergic P1 / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014970:Xanthines / Q000494:pharmacology; D001619:beta-N-Acetylhexosaminidases / Q000378:metabolism
chemical_list: D018076:DNA, Complementary; D015226:Dinucleoside Phosphates; D009363:Neoplasm Proteins; D009636:Norbornanes; D012333:RNA, Messenger; D043684:Receptor, Adenosine A3; D018047:Receptors, Purinergic P1; C099530:WRC 0571; D014970:Xanthines; C086749:BW A522; C084956:N(6)-(3-iodobenzyl)-5'-N-methylcarboxamidoadenosine; C013907:cytidylyl adenosine; D001619:beta-N-Acetylhexosaminidases; D000225:Adenine; D000241:Adenosine; D002118:Calcium