IDENTIFICATION OF A REGULATED NA/K/CL COTRANSPORT SYSTEM IN A DISTAL NEPHRON CELL-LINE

Lack of an adequate cell model has limited investigation of Na/K/Cl cotransporter regulation in the kidney. Using A6 cells, an amphibian distal renal cell line, we observed that 63% of rubidium uptake in confluent A6 monolayers was ouabain-insensitive. Ouabain-insensitive rubidium uptake was inhibited in a dose-dependent fashion by furosemide (IC50 6.6 muM) or bumetanide (IC50 1.7 muM). Kinetic studies confirmed that furosemide-sensitive rubidium uptake had features consistent with cotransporter activity in other cell lines. Futhermore, specific binding of [H-3]bumetanide occurred with a capacity of 8.6 pmol/mg protein and a K(d) of 1.6 muM bumetanide. Finally, furosemide-sensitive rubidium uptake was rapidly regulated by a calcium inophore, the phorbol ester PDBu, forskolin, and adenosine. These data demonstrate an Na/K/Cl cotransport system in the A6 cell which will serve as a useful model for studying cotransporter regulation by endogenous signaling pathways.