Purinergic-induced signaling in C11-MDCK cells inhibits the secretory Na-K-Cl cotransporter

1 Nephrology Group, Research Center L'Hôtel-Dieu de Québec, Department of Medicine, Faculty of Medicine, Laval University, Québec G1R 2J6; 2 Research Centre l'Hôtel-Dieu de Montréal, Centre Hospitalier de l'Université de Montréal, Montréal, Quebec, Canada H2W 1T8; 3 Faculty of Biology, MV Lomonosov Moscow State University, Moscow, Russia; 4 Department of Pharmacology, University of California, San Diego, La Jolla, California 92093; and 5 Department of Physiology, University of Wurzburg, D97070 Wurzburg, Germany Submitted 25 August 2002 ; accepted in final form 4 August 2003 Purinergic inhibition of Na-K-Cl cotransport has been noted in various renal epithelial cells derived from the collecting tubule, including Madin-Darby canine kidney (MDCK) cells. In recent studies, we have observed purinergic inhibition of Na-K-Cl cotransport in C11-MDCK subclones ( -intercalated-like cells). Interestingly, Na-K-Cl cotransport activity was also detected in C7-MDCK subclones (principal-like cells) but was not affected by ATP. In this investigation, we have transfected the human Na-K-Cl cotransporter (huNKCC1) in both C11 and C7 cells to determine whether these differences in NKCC regulation by ATP were due to cell-specific purinoceptor signaling pathways or to cell-specific isoforms/splice variants of the transporter. In both cell lines, we found that endogenous as well as huNKCC1-derived cotransport activity was restricted to the basolateral side. In addition, we were able to show that extracellular application of 100 µM ATP or 100 µM UTP abolished NKCC activity in both mock- and huNKCC1-transfected C11 cells but not in mock- and huNKCC1-transfected C7 cells; in C11 cells, intriguingly, this inhibition was not affected by inhibitors of RNA and protein synthesis and occurred even though expression levels of UTP-sensitive P 2Y2 -, P 2Y4 -, and P 2Y6 -purinoceptors were not different from those observed in C7 cells. These results suggest that C11 cells express an undetermined type of UTP-sensitive P 2 -purinoceptors or a unique P 2Y -purinoceptor-triggered signaling cascade that leads to inhibition of NKCC1. P 2 -purinoceptors; Na-K-Cl cotransporter; C7- and C11-Madin-Darby canine kidney cells Address for reprint requests and other correspondence: P. Isenring, L'Hôtel-Dieu de Québec Research Center, 10 rue McMahon (Rm. 3852), QC, Canada G1R 2J6 (E-mail: paul.isenring{at}crhdq.ulaval.ca ).