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


CONCLUSIONS
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.
Chen
Yi-Ching
YC
National Chung Hsing University, Taichung, Taiwan, China.
Jinn
Tzyy-Rong
TR
Chung
Tse-Yu
TY
Li
Feng-Yin
FY
Fan
Ruey-Jane
RJ
Tzen
Jason Tc
JT
D002118:Calcium / Q000378:metabolism*; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D004365:Drugs, Chinese Herbal / Q000302:isolation & purification / Q000494:pharmacology*; D004721:Endoplasmic Reticulum / Q000378:metabolism; D016166:Free Radical Scavengers / Q000008:administration & dosage / Q000494:pharmacology; D006801:Humans; D006860:Hydrogen Bonding; D008958:Models, Molecular; D009447:Neuroblastoma / Q000378:metabolism; D010042:Ouabain / Q000008:administration & dosage / Q000494:pharmacology; D027561:Salvia miltiorrhiza / Q000737:chemistry*; D019831:Sodium-Calcium Exchanger / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism
D004365:Drugs, Chinese Herbal; D016166:Free Radical Scavengers; D019831:Sodium-Calcium Exchanger; C061091:lithospermate B; D010042:Ouabain; D000254:Sodium-Potassium-Exchanging ATPase; D002118:Calcium