Effect of magnesium lithospermate B on calcium and nitric oxide in endothelial cells upon hypoxia/reoxygenation.

AIM

To investigate the effect of magnesium lithospermate B (MLB) on hypoxia/ reoxygenation (H/R)-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 exposed to hypoxia for 30 min under 95 % N2 and 5 % CO2, then reoxygenation for 30 min under air and 5 % CO2. Cell injury was evaluated by dye exclusion test, superoxide dismutase (SOD) assay, and molondialdehyde (MDA) assay. [Ca2+]i was determined by Fura 2-AM. NO content was examined by a 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 (93.1+/-1.2) % in normoxia to (88+/-3) % in H/R (P < 0.01), and SOD activity was also decreased from (0.24+/-0.07) kNU/L to (0.18+/-0.03) kNU/L in H/R (P >0.05), but MDA production was increased from (1.12+/-0.06) mmol/L in normoxia to (3.78+/-0.03) mmol/L in H/R (P < 0.01) in ECV304 cultured under calcium conditions. MLB 2.5, 5, and 10 mg/L increased cell viability and SOD activity, and inhibited MDA formation in ECV304. H/R increased [Ca2+]i (F340/F380 from 1.65+/-0.16 to 1.89+/-0.28), NO release [from (7.5+/-1.3) micromol/L to (16+/-5) micromol/L], and eNOS mRNA expression, but decreased iNOS mRNA expression in ECV304 (P <0.05). However, it did not affect them under calcium-free conditions. MLB inhibited H/R-induced increases in [Ca2+]i and eNOS mRNA expression, stimulated NO release and iNOS mRNA expression (P <0.05).

CONCLUSION

MLB attenuates H/R-induced cell injury and increases NO release in ECV304. This increase of NO production is possibly associated with preventing cell injury induced by H/R in MLB-treated ECV304.