Development of an HTS assay for Na+, K+-ATPase using nonradioactive rubidium ion uptake.
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.
Gill
Sikander
S
Aurora Biomed, Inc., Vancouver, BC, Canada V6A 1W2. gill@aurorabiomed.com
Gill
Rajwant
R
Wicks
David
D
Despotovski
Sasko
S
Liang
Dong
D
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000379:methods; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D010042:Ouabain / Q000494:pharmacology; D012413:Rubidium / Q000378:metabolism*; D012680:Sensitivity and Specificity; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013054:Spectrophotometry, Atomic / Q000379:methods*
D004791:Enzyme Inhibitors; D010042:Ouabain; D000254:Sodium-Potassium-Exchanging ATPase; D012413:Rubidium