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This Article Full Text Full Text (PDF) All Versions of this Article: 568/3/789    most recent jphysiol.2005.094375v1 Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Akimova, A. O. Articles by Orlov, S. N Search for Related Content PubMed PubMed Citation Articles by Akimova, A. O. Articles by Orlov, S. N

¹ Centre de recherche, Centre hospitalier de l'Université de Montréal (CHUM-Hôtel-Dieu), Montreal, PQ, Canada
² Department of Medicine, University of Chicago, Chicago, IL, USA
³ Department of Pharmacology, University of California, San Diego, CA, USA
⁴ Department of Physiology, University of Wurzburg, Germany

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 0.3 µM. 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_i²⁺ 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.

(Received 8 July 2005; accepted after revision 15 August 2005; first published online 18 August 2005)
Corresponding author S. N. Orlov: Centre de recherche, CHUM – Hôtel-Dieu, 3850 rue St-Urbain, Montréal, Quebec H2W 1T7, Canada. Email: sergei.n.orlov{at}umontreal.ca

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