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+.