Cl−secretion in ATP-treated renal epithelial C7–MDCK cells is mediated by activation of P2Y1 receptors, phospholipase A2 and protein kinase A

doi:10.1113/jphysiol.2005.094375

Cl⁻secretion in ATP-treated renal epithelial C7–MDCK cells is mediated by activation of P_2Y1 receptors, phospholipase A₂ and protein kinase A

^¹Centre 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

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

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 ∼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.