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 comm only used model of the mammalian distal section of the nephron, adenosine A (1) and A(2A) receptor activation modulates sodium and chloride transport a nd intracellular pH (Casavola et al., 1997). Here we show that apical addit ion of the A(3) receptor-selective agonist, 2-chloro-N-6-(3-iodobenzyl)-ade nosine-5' -methyluronamide (Cl-IB-MECA) stimulated a chloride secretion tha t was mediated by calcium- and cAMP-regulated channels. Moreover, in single cell measurements using the fluorescent dye Fura 2-AM, Cl-IB-MECA caused a n increase in Ca2+ influx. The agonist-induced rise in [Ca2+](i), was signi ficantly inhibited by the selective adenosine A(3) receptor antagonists, 2, 3-diethyl-4,5-dipropyl-6-phenylpyridine-3-thio-carboxylate-5-carboxylate (M RS 1523) and 3-ethyl 5-benzyl 2-methyl-6-phenyl-4-phenylethynyl-1,4-(+/-)di hydropyridine-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 [Ca2+](i) by interacting exclusively with A(3) receptors. Cl-IB-MECA-elici ted Ca2+ entry was not significantly inhibited by per tussis toxin pretreat ment while being stimulated by cholera toxin preincubation or by raising ce llular cAMP levels with forskolin or rolipram. Preincubation with the prote in kinase A inhibitor, H89, blunted the Cl-IB-MECA-elicited [Ca2+](i) respo nse. Moreover, Cl-IB-MECA elicited an increase in cAMP production that was inhibited only by an A(3) receptor antagonist. Altogether, these data sugge st that in A(6) cells a G(s)/protein kinase A pathway is involved in the A( 3) receptor-dependent increase in calcium entry.