Canine mast cell adenosine receptors: Cloning and expression of the A(3) receptor and evidence that degranulation is mediated by the A(2B) receptor

We cloned and characterized the canine A(3) adenosine receptor (AR) and examined AR-induced degranulation of the BR line of canine mastocytoma cells. Canine A(3)AR transcript is found predominantly in spleen, lung, liver, and testes and encodes a 314-amino acid heptahelical receptor. I-125-N-6-Aminobenzyladenosine binds to two affinity states of canine A(3)AR with K-D 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 A(3)AR. Although canine BR mastocytoma cells contain A(1)AR, A(2B)AR, and A(3)AR, degranulation seems to be mediated primarily ly A(2B)ARs stimulated by the nonselective agonist 5'-N-ethylcarboxamidoadenosine (NECA) but not by the A(3)-selective agonist N-6-(3-iodobenzyl)adenosine-5'-N-methyl- carboxamide. NECA-stimulated degranulation is not prevented by pertussis toxin and is blocked by enprofylline (K-l = 7 mu M), an antiasthmatic xanthine with low affinity (K-l > 100 mu M) for A(1)AR, A(2A)AR, and A(3)AR. NECA increases canine mastocytoma cell cAMP, Ca2+, and inositol trisphosphate levels; these responses are antagonized half-maximally by 7-15 mu M enprofylline. The results suggest that (i) the cloned canine A(3)AR is structurally and pharmacologically more similar to human than to rat A(3)AR; (ii) the A(2B)AR, and not the A(1)AR or A(3)AR, is principally responsible for adenosine-mediated degranulation of canine BR mastocytoma cells; and (iii) the BR cell A(2B)AR couples to both Ca2+ mobilization and cAMP accumulation. Although A(2B) receptors play a major role in the regulation of BR mast cell degranulation, multiple AR subtypes and G proteins may influence mast cell functions.