Fig. 4.

Effect of different ouabain concentration on Na-K-ATPase-mediated ⁸⁶Rb uptake in kidney proximal tubule cells. A: human kidney cells (HKC-11 or HK-2) were treated for 15 min with different concentrations of ouabain, and Na-K-ATPase-mediated ⁸⁶Rb uptake was measured as described in experimental procedures. Values are means ± SE from 6 independent experiments performed in triplicate. Of note, all ⁸⁶Rb experiments were performed in the presence of a sodium ionophore (monensin; 5 μM) such that the uptake was measured at V_max for sodium. *P < 0.05 by 1-way ANOVA followed by Bonferroni analysis (GraphPad Prizm). B: cells were treated with 5 μM monensin for the indicated time and intracellular sodium was measured as described in experimental procedures. Each bar represents data from 6 individual experiments as fold-change from 0 min (means ± SE). *P < 0.05 by 1-way ANOVA followed by Bonferroni analysis (GraphPad Prizm). C: cells were treated for 15 min with 10 pM ouabain, and crude membranes were prepared as described in experimental procedures. NHE-1 expression was determined by immunoblotting as described in experimental procedures. The nitrocellulose membrane was cut below the 75-kDa marker, the top portion (above 75 kDa) was probed with anti-NHE-1 antibodies, and the bottom portion (below 75 kDa) was probed with anti-actin antibodies. A representative immunoblot from 3 individual experiments is shown.