title: Cl- secretion in ATP-treated renal epithelial C7-MDCK cells is mediated by activation of P 2Y1 receptors, phospholipase A2 and protein kinase A.
abstract: This study examines the mechanism of P 2Y-induced Cl- secretion in monolayers of C7-Madin-Darby canine kidney (MDCK) cells triggered by basolateral application of ATP and measured as transcellular short current (I(SC)). Both ATP-induced arachidonic acid (AA) synthesis and I(SC) in ATP-treated cells were abolished by the phosholipase A2 (PLA2) inhibitor, AACOCF3. The cyclo-oxygenase inhibitor indomethacin decreased I(SC) and cAMP production in ATP-treated cells with an IC50 of approximately 0.3 microm. ATP led to rapid activation of cAMP-dependent protein kinase A (PKA), as estimated by phosphorylation of a vasodilator-stimulated phosphoprotein. PKA activity and I(SC) evoked by ATP, as well as by prostaglandin E1 (PGE1), were diminished in the presence of the PKA inhibitor H-89 or an adenovirus-mediated expression of PKA-inhibitor protein, PKI. In contrast, indomethacin completely blocked the increment of PKA and I(SC) triggered by ATP and AA, but did not affect PKA activation and I(SC) detected with PGE1. The kinetics of [Ca2+]i elevation in ATP- and thapsigargin-treated cells were similar and suppressed by the Ca(2+)i chelator BAPTA. Neither baseline nor maximal increment of ATP-induced I(SC) was affected by thapsigargin and BAPTA. Real-time PCR showed that C7 cells express more mRNA for P 2Y1 and P 2Y2 than for other P 2Y receptor subtypes. The rank order of potency (2MeSATP > ATP > ADP >> UTP) indicates that P 2Y1 rather than P 2Y2 receptors contribute to PKA and I(SC) activation. Viewed collectively, these data show that Cl- secretion in C7-MDCK monolayers treated with basolateral ATP is triggered by P 2Y1 receptors and is mediated by subsequent [Ca2+]i-independent activation of PLA2 and PKA.
authors.affiliation: Centre de recherche, Centre hospitalier de l'Université de Montréal (CHUM-Hôtel-Dieu), Montreal, PQ, Canada.
authors.forename: A Olga
authors.initials: AO
authors.lastname: Akimova
authors.forename: Nathalie
authors.initials: N
authors.lastname: Bourcier
authors.forename: Sebastien
authors.initials: S
authors.lastname: Taurin
authors.forename: Richard A
authors.initials: RA
authors.lastname: Bundey
authors.forename: Konrad
authors.initials: K
authors.lastname: Grygorczyk
authors.forename: Michael
authors.initials: M
authors.lastname: Gekle
authors.forename: Paul A
authors.initials: PA
authors.lastname: Insel
authors.forename: Nickolai O
authors.initials: NO
authors.lastname: Dulin
authors.forename: Sergei N
authors.initials: SN
authors.lastname: Orlov
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000818:Animals; D002460:Cell Line; D002713:Chlorine / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004285:Dogs; D004789:Enzyme Activation / Q000187:drug effects; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism*; D007668:Kidney / Q000187:drug effects / Q000378:metabolism*; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D018048:Receptors, Purinergic P2 / Q000378:metabolism*; D058472:Receptors, Purinergic P2Y1
chemical_list: D018048:Receptors, Purinergic P2; D058472:Receptors, Purinergic P2Y1; D002713:Chlorine; D000255:Adenosine Triphosphate; D017868:Cyclic AMP-Dependent Protein Kinases; D010741:Phospholipases A; D054467:Phospholipases A2