Magnesium lithospermate B extracted from Salvia miltiorrhiza elevates intracellular Ca(2+) level in SH-SY5Y cells

Abstract

Aim: To examine if magnesium lithospermate B (MLB), a potent inhibitor of Na(+)/K(+)-ATPase, leads to the elevation of intracellular Ca(2+) level as observed in cells treated with cardiac glycosides.

Methods: Viability of SH-SY5Y neuroblastoma cells treated with various concentrations of ouabain or MLB was measured. Intracellular Ca(2+) levels were visualized using Fluo4-AM (fluorescent dye) when cells were treated with ouabain or MLB in the presence or absence of KB-R7943 (Na(+)/Ca(2+) exchanger inhibitor) and 2-APB (IP(3) receptor antagonist). Molecular modeling was conducted for the docking of ouabain or MLB to Na(+)/K(+)-ATPase. Changes of cell body and dendrite morphology were monitored under a microscope.

Results: severe toxicity was observed in cells treated with ouabain of concentration higher than 1 micromol/L for 24 h while no apparent toxicity was observed in those treated with MLB. Intracellular Ca(2+) levels were substantially elevated by MLB (1 micromol/L) and ouabain (1 micromol/L) in similar patterns, and significantly reduced in the presence of KB-R7943 (10 micromol/L) or 2-APB (100 micromol/L). Equivalent interaction with the binding cavity of Na(+)/K(+)-ATPase was simulated for ouabain and MLB by forming five hydrogen bonds, respectively. Treatment of ouabain (1 micromol/L), but not MLB (1 mumol/L), induced dendritic shrink of SH-SY5Y cells.

Conclusion: Comparable to ouabain, MLB leads to the elevation of intracellular Ca(2+) level presumably via the same mechanism by inhibiting Na(+)/K(+)-ATPase. The elevated Ca(2+) levels seem to be supplied by Ca(2+) influx through the reversed mode of the Na(+)/Ca(2+) exchanger and intracellular release from endoplasmic reticulum.

Figure 1

Effects of MLB and ouabain on viability of SH-SY5Y cells. SH-SY5Y cells were treated with various concentrations of MLB and ouabain for 5, 10, 30, 60 min, and 24 h. Viability was measured by MTT assay. Data are mean±SEM (n=3).

Figure 2

Fluctuation of intracellular Ca²⁺ levels of SH-SY5Y cells treated with MLB and ouabain. SH-SY5Y cells were loaded with Fluo4-AM prior to incubation with 1 μmol/L of MLB or ouabain. Intensity of fluorescence was collected and calculated at different time intervals for 30 min (A). Each point is representative for 40 ROIs of time-lapse images in 5 independent experiments. Serial images of cells treated with MLB and ouabain for 0.5, 7, 15, and 30 min were captured to display the fluctuation of intracellular Ca²⁺ levels (B). Scale bar represents 20 μm.

Figure 3

Effects of KB-R7943 on intracellular Ca²⁺ levels of SH-SY5Y cells elevated by MLB and ouabain. Similar Ca²⁺ fluorescence imaging was executed as described in Figure 2 except that cells were treated with KB-R7943 for 5 min before the loading of MLB and ouabain. Data represent mean±SEM (n=3).

Figure 4

Effect of 2-APB on intracellular Ca²⁺ levels of SH-SY5Y cells elevated by MLB and ouabain. Similar Ca²⁺ fluorescence imaging was executed as described in Figure 2 except that cells were treated with 2-APB for 15 min before the loading of MLB and ouabain. Data represent mean±SEM (n=3).

Figure 5

Detailed molecular interactions between the extracellular binding pocket of Na⁺/K⁺-ATPase and ouabain or MLB. (Upper panels) Chemical structures of ouabain and MLB. (Middle panels) Modeling of ouabain and MLB binding to the extracellular pocket of Na⁺/K⁺-ATPase α subunit. The amino acid residues around the binding pocket of Na⁺/K⁺-ATPase are shown in ribbon structure, and ouabain and MLB in scaled ball and stick. (Lower panels) The amino acid residues of Na⁺/K⁺-ATPase close to ouabain or MLB are shown in wireframe, and the structures of ouabain and MLB in scaled ball and stick. Green box or oval represents one or two hydrogen bonds formed between Na⁺/K⁺-ATPase and ouabain or MLB.

Figure 6

Examination of cell and dendrite volumes after treatment of MLB and ouabain. Changes of cell and dendrite volumes were measured after treated with 1 μmol/L of MLB and ouabain for 30 min. Dendrites indicated by arrows were selected from the examined cells (box areas in left panels) and shown in enlarged photos (right panels). Relative volume (cell or dendrite volume at 30 min over its volume at 0 min) was calculated and shown at the bottom figure. Data represent mean±SEM (n=3). ^bP<0.05 vs control group. Scale bar represents 20 μm.