Inhibition of Na+ and Ca2+ reabsorption by P-2u purinoceptors requires PKC but not Ca2+ signaling

Rabbit connecting tubule and cortical collecting duct cells were isolated by immunodissection and cultured to confluence on permeable filters and on glass coverslips. Extracellular ATP dose-dependently reduced transcellular Na+ and Ca2+ transport (half-maximal inhibitory concentration, IC50, of 0.5 +/- 0.2 and 3.2 +/- 0.5 mu M), with a maximal inhibition of 57 +/- 5 and 43 +/- 4%, respectively. Purinergic receptor agonists inhibited transport with the following rank order of potency: UTP = ATP > ADP; this suggests involvement of P-2u purinoceptors. ATP also caused a dose-dependent (50% effective dose, EC(50), of 1.5 +/- 0.2 mu M) transient increase in intracellular Ca2+ concentration ([Ca2+](i)), which decreased to a sustained elevated level. In the absence of extracellular Ca2+, a similar Ca2+ transient occurred, but the sustained response was abolished. Preloading the cells with the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) completely prevented the ATP-induced Ca2+ transients, but not the ATP-induced inhibition of Na+ and Ca2+ absorption. Activation of protein kinase C (PKC) by the cell-permeable diacylglycerol analogue, 1,2-dioctanoyl-sn-glycerol, mimicked ATP-induced inhibition of Na+ and Ca2+ absorption. The inhibitory effects of ATP were no longer observed after culturing cells in the presence of phorbol eater (12-O-tetradecanoylphorbol-13-acetate) for 5 days, which resulted in downregulation of cellular PKC activity.