A new chromatographic method for measurement of rubidium transport activities in cultured bovine retinal pigment epithelial cells

A new method for measuring cellular rubidium (Rb⁺) uptake activities based on cation chromatography was developed and compared with the standard technique, uptake of the radioisotope ⁸⁶Rb⁺, using cultured bovine retinal pigment epithelial (RPE) cells. The Rb⁺ response was strictly linear from 0.25 nmol (detection limit) to 25 nmol. The Na⁺/K⁺-ATPase inhibitor ouabain inhibited Rb⁺ uptake with IC₅₀ values of 128.7 ± 23.5 nM (n = 8; radioactive method) and 56.6 ± 9.3 nM (n = 9; non-radioactive method, p < 0.01). The latter value is identical to the IC₅₀ value of 54.4 ± 16.2 nM (n = 3) for ouabain binding to the intact RPE cells. Ouabain and bumetanide, an inhibitor of the Na⁺/K⁺/Cl⁻ cotransporter, each inhibited Rb⁺ uptake maximally by 66 and 30%, respectively. This new technique allows sensitive measurement of intracellular Rb⁺, as well as K⁺ and Na⁺, and, thus, should prove useful for studying the effects of pharmacologic agents and simulated disease conditions on cation transport and cation balance in RPE and other cell types.