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J. Biol. Chem., Vol. 281, Issue 42, 31317-31325, October 20, 2006

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Transient Activation and Delayed Inhibition of Na⁺,K⁺,Cl^– Cotransport in ATP-treated C11-MDCK Cells Involve Distinct P2Y Receptor Subtypes and Signaling Mechanisms^*

Olga A. Akimova, Alexandra Grygorczyk, Richard A. Bundey, Nathalie Bourcier, Michael Gekle^¶, Paul A. Insel^||, and Sergei N. Orlov¹

From the Centre de Recherche, Centre Hospitalier de l'Université de Montréal-Technopôle Angus, Montreal, Quebec H1W 4A4, Canada, the Departments of Pharmacology and ^||Medicine, University of California, San Diego, La Jolla, California 92093, and the ^¶Department of Physiology, University of Wurzburg, 97070 Wurzburg, Germany

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 UTP >> 2-(methylthio)-ATP (2MeSATP); adenosine 5'-[-thio]diphosphate (ADPS) 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 > ADPS > 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 ADPS 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.

Received for publication, March 6, 2006 , and in revised form, July 10, 2006.

^* This work was supported by grants from the Kidney Foundation of Canada (to S. N. O.) and National Institutes of Health Grant GM66232 (to P. A. I.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Centre de Recherche, CHUM-Technopôle Angus, 2901 Rachel Es Rm. 313, Montreal, Quebec H1W 4A4, Canada. Tel.: 514-890-8000 (ext. 23615); Fax: 514-412-7638; E-mail: sergei.n.orlov{at}umontreal.ca.

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