title: Inhibition of Na+ and Ca2+ reabsorption by P2u purinoceptors requires PKC but not Ca2+ signaling.
abstract: 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 microM), 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 P2u purinoceptors. ATP also caused a dose-dependent (50% effective dose, EC50, of 1.5 +/- 0.2 microM) 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-en-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 ester (12-O-tetradecanoylphorbol-13-acetate) for 5 days, which resulted in downregulation of cellular PKC activity.
authors.affiliation: Department of Cell Physiology, University of Nijmegen, The Netherlands.
authors.forename: H P
authors.initials: HP
authors.lastname: Koster
authors.forename: A
authors.initials: A
authors.lastname: Hartog
authors.forename: C H
authors.initials: CH
authors.lastname: van Os
authors.forename: R J
authors.initials: RJ
authors.lastname: Bindels
mesh_terms: D000042:Absorption / Q000187:drug effects; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D004789:Enzyme Activation; D005110:Extracellular Space / Q000378:metabolism; D007425:Intracellular Membranes / Q000378:metabolism; D007685:Kidney Tubules, Collecting / Q000166:cytology / Q000378:metabolism*; D009994:Osmolar Concentration; D011493:Protein Kinase C / Q000502:physiology*; D011817:Rabbits; D011983:Receptors, Purinergic / Q000502:physiology*; D015398:Signal Transduction; D012964:Sodium / Q000037:antagonists & inhibitors*
chemical_list: D011983:Receptors, Purinergic; D000255:Adenosine Triphosphate; D012964:Sodium; D011493:Protein Kinase C; D002118:Calcium