Identification of hydroxyxanthones as Na/K-ATPase ligands

Abstract

We have screened a chemical library and identified several novel structures of Na/K-ATPase inhibitors. One group of these inhibitors belongs to polyphenolic xanthone derivatives. Functional characterization reveals the following properties of this group of inhibitors. First, like ouabain, they are potent inhibitors of the purified Na/K-ATPase. Second, their effects on the Na/K-ATPase depend on the number and position of phenolic groups. Methylation of these phenolic groups reduces the inhibitory effect. Third, further characterization of the most potent xanthone derivative, MB7 (3,4,5,6-tetrahydroxyxanthone), reveals that it does not change either Na(+) or ATP affinity of the enzyme. Finally, unlike that of ouabain, the inhibitory effect of MB7 on Na/K-ATPase is not antagonized by K(+). Moreover, MB7 does not activate the receptor Na/K-ATPase/Src complex and fails to stimulate protein kinase cascades in cultured cells. Thus, we have identified a group of novel Na/K-ATPase ligands that can inhibit the pumping function without stimulating the signaling function of Na/K-ATPase.

Fig. 1.

A, concentration curves of ouabain, MB7, and quercetin on the Na/K-ATPase. The purified Na/K-ATPase was incubated with different concentrations of compounds for 15 min, then assayed for ouabain-sensitive ATPase activity as described under Materials and Methods. The data are combined from three to five separate experiments and are presented as mean ± S.E. The IC₅₀ for ouabain and MB7 are 1 ± 0.1 and 1.6 ± 0.1 μM, respectively. B, the chemical structures of xanthone (left) and quercetin (right).

Fig. 2.

Effects of MB7 on Na⁺, K⁺, and ATP dependence. Na/K-ATPase activity was measured as in Fig. 1 as a function of Na⁺ (A), K⁺ (B), or ATP (C) concentration. The data are combined from four to six separate experiments and are plotted as mean ± S.E. MB7 was used at 1.5 μM. The apparent K_m values for Na⁺ are 11 ± 1 and 9 ± 1 mM for control and MB7-treated Na/K-ATPase, respectively. The apparent K_m values for K⁺ are 0.6 ± 0.1 and 0.5 ± 0.1 mM for control and MB7, respectively. The K_m values for ATP are 0.25 ± 0.02 and 0.22 ± 0.02 mM for control and MB7-treated Na/K-ATPase, respectively.

Fig. 3.

Effects of MB7 and ouabain on the receptor Na/K-ATPase/Src complex. The purified Na/K-ATPase and purified Src were incubated in the presence of either ouabain (1 μM) or MB7 (1 and 10 μM) as indicated for 15 min and assayed for Src activation as described under Materials and Methods. Values are mean ± S.E. of three independent experiments. **, p < 0.01 compared with control.

Fig. 4.

Effects of MB7 and ouabain on Src and ERKs. A, A549 cells were treated with ouabain or MB7 for 10 min, and cell lysates (50 μg/lane) were separated by SDS polyacrylamide gel electrophoresis and analyzed for Src activation as in Fig. 3. The values are mean ± S.E. from four to six separate experiments. *, p < 0.05 versus control. B, LLC-PK1 cells were treated with MB7 or ouabain for 10 min and immunostained with the ERK/MAPK (phospho-Thr202/Tyr204) phosphorylation/translocation cell-based assay kit (Cayman Chemical) according to the manufacturer's instructions. The images were collected as described under Materials and Methods. Top, representative images from three experiments are shown. Scale bar, 50 μm. Bottom, the quantitative data of phospho-ERK fluorescence intensity were collected from 40 different fields in three independent experiments and expressed as mean ± S.E. *, p < 0.05 versus control.