Development of an HTS Assay for Na⁺, K⁺-ATPase Using Nonradioactive Rubidium Ion Uptake

Abstract

A high-throughput screening (HTS) assay was developed for the Na(+),K(+)-ATPase channel in order to study rubidium uptake as a measure of the functional activity and modulation of this exchanger. The assay uses elemental rubidium as a tracer for K(+) ions. Three cell lines were used to study the exchanger, and the assay was performed in a 96-well microtiter plate format. Rb(+) uptake was carried by the CHO-K1 cells at 37 degrees C; the maximum ion influx was at 80 min of incubation of the cell line in the medium containing 5.4 mM RbCl. The cells were incubated in Rb(+) uptake buffer (5.4 mM) and with the pump blocker ouabain for 1, 2, and 3 h, respectively. A complete block of the Rb(+) uptake was observed with a 5 mM concentration of ouabain for all the three time intervals. The ouabain 50% inhibitory concentration (IC(50)) value for CHO-K1 cell line ATPase was observed to be 298 microM after 3 h of incubation. In addition, IC(50) values of 94 and 89 microM were observed at 30 min of incubation, indicating that the protocol shows reproducible results. A Z' factor higher than 0.7 was observed in the assays. These studies extend the profile of Na(+),K(+)-ATPases and demonstrate the feasibility of this HTS assay system to screen for compounds that pharmacologically modulate the function of Na(+),K(+)-ATPase.

Enhancing the anticancer properties of cardiac glycosides by neoglycorandomization

“…To assess how these structural modifications affect the ability of library members to inhibit Na ϩ ͞K ϩ -ATPases, a fundamental activity of cardiac glycosides (17), library hits 5␤, 15␤, 23␤, 27␤, 33␤, 40␤, and digitoxin (1) were submitted to a nonradioactive rubidium uptake assay to gauge Na ϩ ͞K ϩ -ATPase inhibition in both HEK-239 human embryonic kidney cells and CHO-K1 hamster ovary cells (30). In HEK-239 cells, digitoxin displayed an IC 50 of 75.4 Ϯ 0.5 M, whereas none of the library hits showed 50% inhibition even at the highest concentration tested (300 M for 5␤, 15␤, 23␤, and 33␤; 200 M for 27␤ and 40␤).…”

Elucidating the contribution of the elemental composition of fetal calf serum to antigenic expression of primary human umbilical-vein endothelial cells in vitro

“…Rubidium has an undefined role although has been demonstrated to enter cells using the same protein pump system used to transport potassium ions. The concentrations of this element were found to be significantly higher in sera from donor 7 (Fig.3.A.) [35].…”

Regulation of cough by neuronal Na+–K+ ATPases

“…Pharmacological characterization of sodium pumps and their inhibitors relies primarily on ATP hydrolysis assays of sodium pump isozymes dissociated and enriched from cell membranes, although other assays of pump function have been described [5,47,57,66,67]. But there are additional mechanisms for modulation of sodium pump activity that may occur in intact cells.…”