title: Transient activation and delayed inhibition of Na+,K+,Cl- cotransport in ATP-treated C11-MDCK cells involve distinct P2Y receptor subtypes and signaling mechanisms.
abstract: In C11-MDCK cells, which resemble intercalated cells from collecting ducts of the canine kidney, P2Y agonists promote transient activation of the Na+,K+,Cl- cotransporter (NKCC), followed by its sustained inhibition. We designed this study to identify P2Y receptor subtypes involved in dual regulation of this carrier. Real time polymerase chain reaction analysis demonstrated that C11-MDCK cells express abundant P2Y1 and P2Y2 mRNA compared with that of other P2Y receptor subtypes. The rank order of potency of agents (ATP approximately UTP >> 2-(methylthio)-ATP (2MeSATP); adenosine 5'-[beta-thio]diphosphate (ADPbetaS) inactive) indicated that P2Y2 rather than P2Y1 receptors mediate a 3-4-fold activation of NKCC within the first 5-10 min of nucleotide addition. NKCC activation in ATP-treated cells was abolished by the intracellular calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, calmodulin (CaM) antagonists trifluoroperazine and W-7, and KN-62, an inhibitor of Ca2+/CaM-dependent protein kinase II. By contrast with the transient activation, 30-min incubation with nucleotides produced up to 4-5-fold inhibition of NKCC, and this inhibition exhibited a rank order of potency (2MeSATP > ADPbetaS > ATP >> UTP) typical of P2Y1 receptors. Unlike the early response, delayed inhibition of NKCC occurred in 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-loaded cells and was completely abolished by the P2Y1 antagonists MRS2179 and MRS2500. Transient activation and delayed inhibition of NKCC in C11 cell monolayers were observed after the addition of ATP to mucosal and serosal solutions, respectively. NKCC inhibition triggered by basolateral application of ADPbetaS was abolished by MRS2500. Our results thus show that transient activation and delayed inhibition of NKCC in ATP-treated C11-MDCK cells is mediated by Ca2+/CaM-dependent protein kinase II- and Ca2+-independent signaling triggered by apical P2Y2 and basolateral P2Y1 receptors, respectively.
authors.affiliation: Centre de Recherche, Centre Hospitalier de l'Université de Montréal-Technopôle Angus, Montreal, Quebec H1W 4A4, Canada.
authors.forename: Olga A
authors.initials: OA
authors.lastname: Akimova
authors.forename: Alexandra
authors.initials: A
authors.lastname: Grygorczyk
authors.forename: Richard A
authors.initials: RA
authors.lastname: Bundey
authors.forename: Nathalie
authors.initials: N
authors.lastname: Bourcier
authors.forename: Michael
authors.initials: M
authors.lastname: Gekle
authors.forename: Paul A
authors.initials: PA
authors.lastname: Insel
authors.forename: Sergei N
authors.initials: SN
authors.lastname: Orlov
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D000818:Animals; D001692:Biological Transport; D002147:Calmodulin / Q000378:metabolism*; D002713:Chlorine / Q000737:chemistry; D003838:Deoxyadenine Nucleotides / Q000494:pharmacology; D004285:Dogs; D004789:Enzyme Activation; D005786:Gene Expression Regulation; D007700:Kinetics; D011188:Potassium / Q000378:metabolism; D018048:Receptors, Purinergic P2 / Q000378:metabolism / Q000502:physiology*; D028021:Sodium-Potassium-Chloride Symporters / Q000502:physiology*; D064506:Solute Carrier Family 12, Member 2
chemical_list: C495475:2-iodo-N(6)-methyl-(N)-methanocarba-2'-deoxyadenosine-3',5'-bisphosphate; D002147:Calmodulin; D003838:Deoxyadenine Nucleotides; D018048:Receptors, Purinergic P2; D028021:Sodium-Potassium-Chloride Symporters; D064506:Solute Carrier Family 12, Member 2; D002713:Chlorine; D000255:Adenosine Triphosphate; D011188:Potassium