诱导型一氧化氮合酶在17β-雌二醇诱导的血管平滑肌细胞周期阻滞中的作用

利用大鼠血管平滑肌细胞(vascular smooth muscle cells,VSMC)作为模型,观察17β-雌二醇(17β-estradiol,E2)对VSMC诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)活性和蛋白表达的影响,并探讨其在内皮素-1(endothlin 1,ET-1)刺激的VSMC周期循环中的作用。检测指标包括同位素法测定iNOS的活性,免疫印迹法(western blot)检测iNOS蛋白表达,流式细胞仪检测细胞周期,观察一氧化氮合酶(nitric oxide synthase,NOS)抑制剂N^G-硝基左旋精氨酸甲酯(N^G-nitro—L—arginine methylester,L—NAME)对E2抑制VSMC细胞周期的影响。结果显示,E2明显增加iNOS的活性和蛋白表达,在30min和12h时能诱导VSMC的iNOS活性明显增加,而60min和24h时VSMC的iNOS活性与对照组无显著差异,不呈明显浓度依赖性,雌激素受体(estrogen receptor,ER)拮抗剂Tamoxifen和L—NAME能明显抑制E2诱导的VSMC iNOS活性增加;E2增加VSMC的iNOS蛋白表达的作用在3h时起效,12h达高峰,以后逐渐下降,呈浓度依赖性,Tamoxifen能明显抑制马诱导的VSMC iNOS蛋白表达;E2明显抑制ET-1诱导的S期细胞百分比和G2 S／G1增加,使VSMC在G1期发生细胞周期阻滞,这些作用可被预先给予L—NAME所明显减轻。上述结果提示,E2使ET—l刺激的VSMC细胞周期循环在G1期发生阻滞与增加VSMC iNOS活性有关,该作用至少部分通过ER介导。

Role of inducible nitric oxide synthase in the vascular smooth muscle cells cycle arrest induced by 17 beta-estradiol

Clinical epidemiologic data and animal experimental studies regard estrogen as being protective against the development of cardiovascular diseases. The mechanisms by which estrogen affects the development of vascular diseases are not clear. Recent studies demonstrated that the cardiovascular protective effects of estrogen are closely related to nitric oxide (NO) pathway. Our previous study proved that estrogen inhibited the proliferation and oncogene expression of vascular smooth muscle cells (VSMCs) induced by endothlin 1 (ET-1) and serum,this effect was mediated by NO release. In the present study, we investigated the role of inducible nitric oxide synthase (iNOS) in the VSMCs cycle arrest induced by 17 beta-estradiol (E(2)). The effects of E(2) on iNOS activity and protein expression in cultured rat VSMCs and the influence of NOS inhibitor N(G)-nitro-L-arginine methylester (L-NAME) on the inhibitory effect of E(2) on cell cycle were investigated. NOS assay kit was used to measure the activity of iNOS and protein expression of iNOS was determined by Western-blot. Cell cycle analysis was accessed by flow cytometry. The results obtained showed that E(2) increased iNOS activity of VSMCs but not in a dose-dependent manner. E(2) 10 nmol/L increased the iNOS activity of VSMCs distinctly at two time points: 30 min and 12 h.These effects were significantly inhibited by estrogen receptor (ER) antagonist Tamoxifen (0.1 micromol/L) and NOS inhibitor L-NAME (1 micromol/L). E(2) increased iNOS protein expression of VSMCs in a dose-dependent manner. The effect of E(2) on iNOS protein expression of VSMCs started at 3 h, distinctly increased at 12 h and then decreased. Tamoxifen significantly inhibited the E(2)-induced iNOS protein expression of VSMCs. ET-1 increased cell percentage of S phase and G(2)+S/G(1). This effect was inhibited by E(2). L-NAME significantly attenuated the inhibitory effect of E(2) on cell cycle of VSMCs.The results suggest that E(2) induced G(1) arrest of VSMCs, which was associated with an increase in iNOS activity and protein expression of VSMCs. These effects were at least mediated by estrogen receptor partly.