A simple method for evaluation of Rb+ transport and Na(+)-K+ pump stoichiometry in adherent cells.
This report describes a method based on flame photometry for the evaluation of transmembrane Rb+ transport and Na(+)-K+ pump stoichiometry in adherent cells. In monolayers of cultured fibroblasts, the rates of 86Rb+, an isotope widely used as a K+ congener in transport studies, and nonradioactive Rb+ influx were equivalent when measured in the absence and presence of the transport inhibitors ouabain and bumetanide. Ouabain- and bumetanide-sensitive Rb+ fluxes were also equal with the two methods. Flame photometry allowed the simultaneous determination of intracellular [Na+] in the same sample in which Rb was measured. The incubation of human fibroblasts with ouabain for 5 min promoted a significant increase in intracellular [Na+]. Under appropriate experimental conditions, the ratio between the rate of ouabain-promoted increase in intracellular [Na+] and ouabain-sensitive Rb+ influx was 1.4, close to the theoretical value of 1.5 corresponding to a Na(+)-K+ pump stoichiometry of 3 Na+ extruded from the cell in exchange for 2 K+.
Longo
N
N
Department of Pediatrics, Emory University, Atlanta, Georgia 30322.
Griffin
L D
LD
Elsas
L J
LJ
D001693:Biological Transport, Active / Q000187:drug effects; D002034:Bumetanide / Q000494:pharmacology; D002448:Cell Adhesion; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D010042:Ouabain / Q000494:pharmacology; D011865:Radioisotope Dilution Technique; D012413:Rubidium / Q000378:metabolism*; D012414:Rubidium Radioisotopes; D012867:Skin / Q000166:cytology / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*
D012414:Rubidium Radioisotopes; D002034:Bumetanide; D010042:Ouabain; D000254:Sodium-Potassium-Exchanging ATPase; D012413:Rubidium