Magnesium lithospermate B inhibits hypoxia-induced calcium influx and nitric oxide release in endothelial cells.
OBJECTIVE
To investigate the inhibitory effect of magnesium lithospermate B (MLB) on hypoxia-induced elevation of intracellular calcium concentration ([Ca2+]i) and nitric oxide (NO) release in endothelial cells.


METHODS
The cultured human umbilical vein endothelial cells (ECV304) were cultured for 30 min under 95 % N(2) and 5 % CO2. Cell injury was evaluated by dye exclusion test and lactate dehydrogenase (LDH) assay. [Ca2+]i was determined by Fura 2-AM. NO content was examined by the NO assay kit. Endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) mRNA expressions were measured by semi-quantitative RT-PCR.


RESULTS
Cell viability was decreased from (93.0 +/- 2.6) % in normoxia to (85.5 +/- 2.1) % in hypoxia (P < 0.01), and LDH release was increased from (41 +/- 28) U/L in normoxia to (141+/-68) U/L in hypoxia (P < 0.01) in ECV304 cultured under calcium conditions. MLB 5 and 10 mg/L improved cell viability and inhibited LDH leakage in ECV304. In addition, hypoxia increased [Ca2+]i, NO release, and eNOS and iNOS mRNA expressions in ECV304 (P < 0.01). These increases could be inhibited by MLB 5 and 10 mg/L (P < 0.01), but they were unaffected by hypoxia under calcium-free conditions.


CONCLUSIONS
MLB attenuates hypoxia-induced cell injury and inhibits hypoxia-induced increases of [Ca2+]i, NO release, and eNOS and iNOS mRNA expressions in ECV304 in Krebs'solution containing calcium. The decreases of NO production and eNOS mRNA expression are possibly associated with inhibition of extracellular calcium influx in MLB-treated ECV304
Luo
W B
WB
Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
Wang
Y P
YP
D001693:Biological Transport, Active; D002118:Calcium / Q000378:metabolism*; D015687:Cell Hypoxia; D002478:Cells, Cultured; D004365:Drugs, Chinese Herbal / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000378:metabolism*; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D009569:Nitric Oxide / Q000096:biosynthesis* / Q000235:genetics; D019001:Nitric Oxide Synthase / Q000096:biosynthesis / Q000235:genetics; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; D010100:Oxygen / Q000378:metabolism*; D010936:Plant Extracts; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D027561:Salvia miltiorrhiza; D014471:Umbilical Veins / Q000166:cytology
D004365:Drugs, Chinese Herbal; D010936:Plant Extracts; D012333:RNA, Messenger; C061091:lithospermate B; D009569:Nitric Oxide; D007770:L-Lactate Dehydrogenase; C496319:NOS2 protein, human; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; D010100:Oxygen; D002118:Calcium