Extracellular ATP increases [Ca2+]i in distal tubule cells.: II.: Activation of a Ca2+-dependent Cl- conductance

We characterized Cl- conductance activated by extracellular ATP in an immortalized cell line derived from rabbit distal bright convoluted tubule (DC1). I-125(-) efflux experiments showed that ATP increased I-125(-) loss with an EC50 = 3 mu M. Diphenylamine-2-carboxylate (10(-3) M) and NPPB (10(-4) M) abolished the I-125(-) efflux. Preincubation with 10 mu M 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester or 10(-7) M thapsigargin inhibited the effect of ATP. Ionomycin (2 mu M) increased I-125(-) efflux with a time course similar to that of extracellular ATP, suggesting that the response is dependent on the intracellular Ca2+ concentration ([Ca2+](i)). The ATP agonist potency order was ATP greater than or equal to UTP > ATP gamma S. Suramin (500 mu M) inhibited the ATP-induced I-125(-) efflux, consistent with P2 purinoceptors. I-125(-) effluxes from cells grown on permeable filters suggest that ATP induced an apical efflux that was mediated via apical P2 receptors. Whole cell experiments showed that ATP (100 mu M) activated outwardly rectifying Cl- currents in the presence of 8-cyclopentyl-1,3-dipropylxanthine, excluding the involvement of P1 receptors. Ionomycin activated Cl- currents similar to those developed with ATP. These results demonstrate the presence of a purinergic regulatory mechanism involving ATP, apical P2Y2 receptors, and Ca2+ mobilization for apical Cl- conductance in a distal tubule cell line.