Activation of Phosphoinositide Breakdown and Elevation of Intracellular Calcium in a Rat RBL-2H3 Mast Cell Line by Adenosine Analogs: Involvement of A(3)-Adenosine Receptors?

A variety of adenosine analogs activate phosphoinositide breakdown in a rat RBL-2H3 mast cell line. It is presumed that an A(3)-adenosine receptor is involved, since the phosphoinositide response is insensitive to xanthines. However, the very potent A(3)- receptor agonist 2-chloro-N(6)-iodobenzyl-N-methylcarboxamidoadenosine (2-CI-IBMECA) with an EC(50) of 4.1 µM is about twofold less potent (and less efficacious) than N-ethylcarboxamidoadenosine (NECA) with an EC(50) of 2.1 µM. The other agents consisting of N(6)-p-aminophenylethyladenosine (APNEA), N(6)-iodobenzylMECA (IB-MECA), N(6)-R- phenylisopropyladenosine (R-PIA), 2-chloroadenosine, N(6)-benzyladenosine, N(6)- cyclohexyladenosine (CHA), N(6)-cyclohexylNECA (CHNECA), 2-(p- carboxyethylphenyl-ethylaminoNECA (CGS 21680), 1,3-dibutylxanthine 7-riboside-5'-N-methylcarboxamide (DBXRM), adenosine, and 8-bromoadenosine are all nearly equipotent with EC(50) values of 5.5-13.9 µM. The rank order of potencies of the analogs in causing an elevation of intracellular calcium is quite different. The potent A(3) receptor agonists 2-CI-IBMECA and IB-MECA with EC(50) values of 0.07 and 0.11 µM, respectively, are about fourfold more potent than N(6)-cyclohexylNECA and about 15-fold more potent than NECA. The other analogs are comparable or somewhat less potent than NECA, some are less efficacious, and 8-bromoadenosine is inactive. The results suggest that stimulation of phosphoinositide breakdown by adenosine analogs in RBL-2H3 cells as measured by IP(1) accumulation is not predictive of IP(3)-mediated elevations of intracellular calcium. Rank order of potency for the calcium response is consonant with intermediacy of A(3)-adenosine receptors, while the former, as measured by [(3)H]IP(1)-formation, probably reflects contributions from both an A(3)-mediated response and some other mechanism. Combinations of subthreshold concentrations of 2-CI-IBMECA with either the A(1)-selective agonist CHA or the A(2A)-selective agonist CGS 21680 caused a marked stimulation of phosphoinositide breakdown, providing further evidence for dual mechanisms. The selective A(3)-adenosine receptor antagonist 3,6-dichloro-2'-(isopropyloxy)-4'-methylflavone (MRS 1067) inhibits 2-CI-IBMECA- and NECA-elicited elevation of calcium levels, and had differential effects on phosphoinositide breakdown, blocking [(3)H]IP(3) accumulation and either blocking (NECA) or having no effect (2-CI-IBMECA) on [(3)H]IP(1) accumulation.