甲基亚砷酸对内皮型一氧化氮合酶活性的影响

In vitro study on the effect of endothelial nitric oxide synthase activity by monomethylarsonous acid

目的研究甲基亚砷酸(MMAⅢ)对内皮型一氧化氮合酶(eNOS)活性的影响,并初步探讨MMAⅢ抑制eNOS活性的作用机制。方法从体外培养的牛大动脉血管内皮细胞中制备eNOS提取液,单独暴露于MMAⅢ(暴露剂量分别为1、2.5、5、10、15μmol/L)或其他砷化合物(亚砷酸钠、砷酸钠、甲基砷酸或二甲基砷酸,暴露剂量均为10μmol/L);或者联合暴露于MMAⅢ(10μmol/L)和二硫苏糖醇(DTT,100μmol/L)。37℃暴露5min后测定L-[3H]精氨酸产生的L-[3H]瓜氨酸量来反映eNOS活性。结果eNOS活性随着MMAⅢ暴露剂量增加而逐渐降低,MMAⅢ的2.5、5、10、15μmol/L剂量组的eNOS活性分别是对照组的44.4%、23.4%、11.7%和6.6%,均显著低于对照组(p<0.01);MMAⅢ对eNOS活性的50%抑制剂量(IC50)为2.1μmol/L;其他砷化合物10μmol/L暴露对eNOS活性均无明显影响(p>0.05);DTT(100μmol/L)能够部分恢复MMAⅢ暴露引起的eNOS活性降低。结论MMAⅢ对eNOS活性具有显著抑制作用;MMAⅢ抑制eNOS活性很可能与MMAⅢ和eNOS分子的二巯基结构的相互作用有关。

Objective To examine the effect of monomethylarsonous acid （ MMA^Ⅲ ） on endothelial nitric oxide synthase （eNOS） activity and the mechanism of eNOS suppression by MMA^Ⅲ. Methods eNOS enzyme, prepared from cultured bovine aortic endothelial cells, was incubated with various concentrations of MMA^Ⅲ （0 - 15 μmol/L） , other arsenicals （arsenite, arsenate, monomethylarsonate acid or dimethylarsinic acid, 10 μmol/L, respectively ） or MMA^Ⅲ （ 10 μmol/L）/Dithiothreitol （DTT, 100 μmol/L） at 37 ℃ for 5 min. Production of L- [^3H ] c itrulline from L- [ ^3H ] arginine was measured to determine eNOS activity. Results eNOS activity was significantly decreased by MMA^Ⅲ exposure （ p 〈0. 01 ） with an IC50 value of 2.1 μmol/L （44.4%, 23.4%, 11.7% and 6. 6% of control levels by MMAmof2.5, 5, 10 and 15 μmol/L, respectively）. Other arsenicals didnt affect the eNOS activity at 10 μmol/L [ P 〉 0. 05 ）. DTT [ 100 μmol/L） effectively reversed the decrease of eNOS activity caused by MMA^Ⅲ exposure. Conclusions MMA^Ⅲ was a potent inhibitor of eNOS. Modification of vicinal thiols in eNOS may participate in the decreased activity of eNOS by MMA^Ⅲ exposure.