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 microM) or bumetanide (IC50 1.7 microM). Kinetic studies confirmed that furosemide-sensitive rubidium uptake had features consistent with cotransporter activity in other cell lines. Furthermore, specific binding of [3H]bumetanide occurred with a capacity of 8.6 pmol/mg protein and a Kd of 1.6 microM bumetanide. Finally, furosemide-sensitive rubidium uptake was rapidly regulated by a calcium ionophore, 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.
Fan
P Y
PY
Department of Internal Medicine, Duke University Medical Center, Durham, NC 27710.
Haas
M
M
Middleton
J P
JP
D000818:Animals; D002460:Cell Line; D002712:Chlorides / Q000378:metabolism*; D005665:Furosemide / Q000494:pharmacology; D017136:Ion Transport; D007668:Kidney / Q000378:metabolism*; D011188:Potassium / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000032:analysis; D014982:Xenopus laevis
D002712:Chlorides; D005665:Furosemide; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium