Activation of A(3) adenosine receptor induces calcium entry and chloride secretion in A(6) cells.

We have previously demonstrated that in A(6) renal epithelial cells, a commonly used model of the mammalian distal section of the nephron, adenosine A(1) and A(2A) receptor activation modulates sodium and chloride transport and intracellular pH (Casavola et al., 1997). Here we show that apical addition of the A(3) receptor-selective agonist, 2-chloro-N(6)-(3-iodobenzyl)-adenosine-5'-methyluronamide (Cl-IB-MECA) stimulated a chloride secretion that was mediated by calcium- and cAMP-regulated channels. Moreover, in single cell measurements using the fluorescent dye Fura 2-AM, Cl-IB-MECA caused an increase in Ca(2+) influx. The agonist-induced rise in [Ca(2+)](i) was significantly inhibited by the selective adenosine A(3) receptor antagonists, 2,3-diethyl-4, 5-dipropyl-6-phenylpyridine-3-thiocarboxylate-5-carboxylate (MRS 1523) and 3-ethyl 5-benzyl 2-methyl-6-phenyl-4-phenylethynyl-1, 4-(+/-)-dihydropyridine-3,5-dicarboxylate (MRS 1191) but not by antagonists of either A(1) or A(2) receptors supporting the hypothesis that Cl-IB-MECA increases [Ca(2+)](i) by interacting exclusively with A(3) receptors. Cl-IB-MECA-elicited Ca(2+) entry was not significantly inhibited by pertussis toxin pretreatment while being stimulated by cholera toxin preincubation or by raising cellular cAMP levels with forskolin or rolipram. Preincubation with the protein kinase A inhibitor, H89, blunted the Cl-IB-MECA-elicited [Ca(2+)](i) response. Moreover, Cl-IB-MECA elicited an increase in cAMP production that was inhibited only by an A(3) receptor antagonist. Altogether, these data suggest that in A(6) cells a G(s)/protein kinase A pathway is involved in the A(3) receptor-dependent increase in calcium entry.