甲基亚砷酸对内皮型一氧化氮合酶磷酸化影响

李冰; 王毅; 孙贵范

doi:10.11847/zgggws2007-23-01-55

甲基亚砷酸对内皮型一氧化氮合酶磷酸化影响

Study on eNOS phosphorylation induced by monomethylarsonous acid

摘要

摘要: 目的观察无机砷的活性中间产物-甲基亚砷酸(MMAⅢ)对牛主动脉血管内皮细胞(BAEC)的内皮型一氧化氮合酶(eNOS)磷酸化以及活性氧自由基(ROS)产生的影响。方法培养的BAEC分别暴露于MMAⅢ(0.75μmol/L,0~15 min);亚砷酸钠(iAsⅢ,100μmol/L,0~60 min);或N-乙酰半胱氨酸(NAC,1 mmol/L)预处理2 h后,再暴露于MMAⅢ(0.75μmol/L,0~15 min)。收获细胞悬液进行eNOS磷酸化蛋白的western blot分析;应用二氯二乙酰荧光素法(H2DCFDA)观测细胞内的ROS产生。结果BAEC暴露于MMAⅢ(0.75μmol/L)15 min后,eNOSSer1179磷酸化显著增加(P<0.05),NAC预处理(1 mmol/L)能够抑制这一现象;iAsⅢ暴露也能够诱导eNOSSer1179磷酸化(P<0.05),但是需要高浓度和更长的暴露时间(100μmol/L,30 min);MMAⅢ(0.75μmol/L)暴露能够诱导BAEC产生ROS,NAC预处理(1 mmol/L)能够抑制这一现象。结论MMAⅢ暴露能够诱导细胞内产生ROS以及ROS依赖性eNOS磷酸化。

Abstract: ObjectiveTo observe the effects of monomethylarso nous acid(MMAⅢ)on eNOS phosphor ylation and reactive oxygen species(ROS)generation in bovine aortic endothelial cells(BAEC).MethodCultured BAEC was exposed to MMAⅢ(0.75 μmol/L,0~15 min)without or with acetylcysteine(NAC)pretreatment(1 mmol/L,2 h),or iAsⅢ(100 Lmol/L,0~60 min).Western blot was done with the specific antibodies for phospho-eNOS_Ser1179.Intracellular ROS generation was assessed with the fluorescent probe,2',7'-dichlorodihydrofluorescein diacetate(H2DCFDA).ResultsMMAⅢ (0.75 μmol/L)caused a rapid increase in phosphorylated eNOS_Ser1179 at 15 min after exposure(P<0.05).In contrast,up to 100 μmol/L iAsⅢ caused the increase of phosphorylated eNOS_Ser1179 at 30 min(P<0.05).MMAⅢs timulated the ROS production in live cells.Both the MMAⅢ-induced eNOS_Ser1179 phosphory lation and ROS generation were blocked by NAC pretreatment(1 mmol/L,2 h).ConclusionMMAⅢ exposure can induce ROS generation and ROS-dependent eNOS phosphor ylation.

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