INHIBITION OF NA-2U PURINOCEPTORS REQUIRES PKC BUT NOT CA2+ SIGNALING( AND CA2+ REABSORPTION BY P)

Rabbit connecting tubule and cortical collecting duct cells were isola ted by immunodissection and cultured to confluence on permeable filter s and on glass coverslips. Extracellular ATP dose-dependently reduced transcellular Na+ and Ca2+ transport (half-maximal inhibitory concentr ation, IC50, of 0.5 +/- 0.2 and 3.2 +/- 0.5 mu M), with a maximal inhi bition of 57 +/- 5 and 43 +/- 4%, respectively. Purinergic receptor ag onists inhibited transport with the following rank order of potency: U TP = ATP > ADP; this suggests involvement of P-2u purinoceptors. ATP a lso 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 sustai ned response was abolished. Preloading the cells with the Ca2+ chelato r ,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) comp letely prevented the ATP-induced Ca2+ transients, but not the ATP-indu ced inhibition of Na+ and Ca2+ absorption. Activation of protein kinas e C (PKC) by the cell-permeable diacylglycerol analogue, 1,2-dioctanoy l-sn-glycerol, mimicked ATP-induced inhibition of Na+ and Ca2+ absorpt ion. The inhibitory effects of ATP were no longer observed after cultu ring cells in the presence of phorbol eater (12-O-tetradecanoylphorbol -13-acetate) for 5 days, which resulted in downregulation of cellular PKC activity.