Acta Pharmacologica Sinica (2010) 31: 923–929;  doi: 10.1038/aps.2010.102

Yi-ching Chen¹, Tzyy-rong Jinn², Tse-yu ChUnG¹, Feng-yin Li³, Ruey-jane FaN^{4, *}, Jason TC Tzen^{1, 2, 5, *}

¹Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, 40227, Taiwan, China; ²School of Chinese Medicine, China Medical University, Taichung, 40402, Taiwan, China; ³Department of Chemistry and ⁴Institute of Genomics and Bioinformatics, National Chung Hsing University, 40227, Taiwan, China; ⁵Agricultural Biotechnology Research Center, Academia Sinica, Taipei 11529, Taiwan, China

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 µmol/L for 24 h while no apparent toxicity was observed in those treated with MLB.  Intracellular Ca²⁺ levels were substantially elevated by MLB (1 µmol/L) and ouabain (1 µmol/L) in similar patterns, and significantly reduced in the presence of KB-R7943 (10 µmol/L) or 2-APB (100 µmol/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 µmol/L), but not MLB (1 µmol/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.   

Keywords: intracellular calcium; cardiac glycoside; magnesium lithospermate B; Na⁺/K⁺-ATPase; ouabain; SH-SY5Y neuroblastoma cells