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Am J Physiol Cell Physiol 260: C1341-C1346, 1991;
0363-6143/91 $5.00
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AJP - Cell Physiology, Vol 260, Issue 6 C1341-C1346, Copyright © 1991 by American Physiological Society

ARTICLES

A simple method for evaluation of Rb+ transport and Na(+)-K+ pump stoichiometry in adherent cells

N. Longo, L. D. Griffin and L. J. Elsas
Department of Pediatrics, Emory University, Atlanta, Georgia 30322.

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+.


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