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

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 A(2) (PLA(2)) inhibitor, AACOCF(3.) The cyclo-oxygenase inhibitor indomethacin decreased I-SC and cAMP production in ATP-treated cells with an IC50 of similar to 0.3 mu m. 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 E-1 (PGE(1)), 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-i(2+) 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 P2Y, receptors and is mediated by subsequent [Ca2+](i) -independent activation of PLA(2) and PKA.