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.