Magnesium lithospermate B extracted from Salvia miltiorrhiza elevats intracellular Ca2+ level in SH-SY5Y cells

Aim: To examine if magnesium lithospermate B (MLB), a potent inhibitor of Na+/K+-ATPase, leads to the elevation of intracellular Ca2+ 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 Ca2+ 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+/Ca2+ exchanger inhibitor) and 2-APB (IP3 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 mu mol/L for 24 h while no apparent toxicity was observed in those treated with MLB. Intracellular Ca2+ levels were substantially elevated by MLB (1 mu mol/L) and ouabain (1 mu mol/L) in similar patterns, and significantly reduced in the presence of KB-R7943 (10 mu mol/L) or 2-APB (100 mu mol/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 mu mol/L), but not MLB (1 mu mol/L), induced dendritic shrink of SH-SY5Y cells. Conclusion: Comparable to ouabain, MLB leads to the elevation of intracellular Ca2+ level presumably via the same mechanism by inhibiting Na+/K+-ATPase. The elevated Ca2+ levels seem to be supplied by Ca2+ influx through the reversed mode of the Na+/Ca2+ exchanger and intracellular release from endoplasmic reticulum.