环磷酸鸟苷依赖性血管舒张剂对血管平滑肌细胞中诱导型一氧化氮合酶表达的影响：环磷酸腺苷的作用。
Effect of cyclic GMP-dependent vasodilators on the expression of inducible nitric oxide synthase in vascular smooth muscle cells: role of cyclic AMP.
作者信息
Boese M, Busse R, Mülsch A, Schini-Kerth V
机构信息
Zentrum der Physiologie, Klinikum der Johann Wolfgang Goethe Universität, Frankfurt/Main, Germany.
出版信息
Br J Pharmacol. 1996 Oct;119(4):707-15. doi: 10.1111/j.1476-5381.1996.tb15730.x.
Abstract
- In the present study we examined whether interleukin-1 beta (IL-1 beta) increases the activity of adenylyl cyclase in vascular smooth muscle cells and determined its role in the cytokine-induced expression of the inducible nitric oxide synthase (iNOS) and activation of nuclear transcription factor-kappa B (NF-kappa B). In addition the interaction between cyclic AMP- and cyclic GMP-elevating agonists on the IL-1 beta-stimulated expression of iNOS was examined. 2. Exposure of vascular smooth muscle cells to IL-1 beta stimulated the formation of cyclic AMP but not of cyclic GMP. The intracellular level of cyclic AMP reached a maximum within 1 h and then gradually declined over the next 5 h. This IL-1 beta (60 u ml-1)-stimulated formation of cyclic AMP was modest (about 3 fold at 60 u ml-1 for 1 h) compared to that evoked by isoprenaline (about 9 fold at 3 x 10(-6) M for 2 min). 3. The IL-1 beta (60 u ml-1 for 24 h)-stimulated accumulation of nitrite, which was taken as an index of NO production, was concentration-dependently increased by preferential inhibitors of cyclic AMP-dependent phosphodiesterases (rolipram and trequinsin). This effect was reproduced by a specific activator of the cyclic AMP-dependent protein kinase(s) A, Sp-8-CPT-cAMPS (10(-4) M) but was prevented by a specific inhibitor of cyclic AMP-dependent protein kinase(s) A, Rp-8-CPT-cAMPS (10(-4) M). These compounds alone [rolipram (10(-6) M), trequinsin (3 x 10(-6) M) and Sp-8-CPT-cAMPS (10(-4) M)] slightly but significantly increased the release of nitric oxide while Rp-8-CPT-cAMPS elicited no such effect. 4. Inducible NOS protein was expressed in IL-1 beta (30 u ml-1, 24 h)-stimulated smooth muscle cells as assessed by Western blot analysis. The level of iNOS protein was markedly increased in smooth muscle cells which had been exposed to IL-1 beta in combination with either rolipram (3 x 10(-6) M) or Sp-8-CPT-cAMPS (10(-4) M) but was reduced in those exposed to IL-1 beta and Rp-8-CPT-cAMPS (10(-4) M). A weak expression of iNOS protein was found in smooth muscle cells which had been exposed to either Sp-8-CPT-cAMPS or rolipram alone for 24 h while Rp-8-CPT-cAMPS elicited no such effect. 5. Exposure of smooth muscle cells to IL-1 beta (30 u ml-1) for 30 min increased the level of NF-kappa B-DNA complexes in nuclear extracts as detected by electrophoretic mobility shift assay. Similar levels of NF-kappa B-DNA complexes were found in cells which had been exposed to IL-1 beta in combination with either Sp-8-CPT-cAMPS (10(-4) M), trequinsin (10(-6) M) or rolipram (10(-6) M). None of the modulators alone affected the basal level of NF-kappa B binding activity. 6. NO-donors [sodium nitroprusside (SNP) 10(-4) M; dinitrosyl-iron-di-L-cysteine-complex (DNIC), 10(-4) M; 3-morpholino-sydnonimine (SIN-1), 10(-4) M] and atrial natriuretic factor (10(-6) M) significantly increased the IL-1 beta (30 or 60 u ml-1, 24 h)-stimulated expression of iNOS protein and activity as assessed indirectly by the conversion of oxyhaemoglobin to methaemoglobin. In the absence of IL-1 beta, SNP (10(-4) M, 24 h) but not the other cyclic GMP-dependent vasodilators caused a modest expression of iNOS protein. No such effect was found in smooth muscle cells exposed to SNP in combination with Rp-8-CPT-cAMPS (10(-4) M) while an increased level of iNOS protein was found in those exposed to SNP in combination with either Sp-8-CPT-cAMPS (10(-4) M) or rolipram (3 x 10(-6) M). 7. Exposure of vascular smooth muscle cells to either S-nitroso-L-cysteine (Cys-SNO, 10(-4) M), SNP (10(-4) M) or SIN-1 (10(-4) M) for 35 min affected minimally the basal activation of NF-kappa B but abolished that evoked by IL-1 beta (30 u ml-1 added during the last 30 min). However, addition of Cys-SNO following the stimulation with IL-1 beta (during the last 5 min of the 30 min exposure period) reduced the level of NF-kappa B-DNA complexes only slightly. 8. These data indicate that the cyclic AMP-dependent pathway plays a decisi
摘要
- 在本研究中，我们检测了白细胞介素-1β（IL-1β）是否会增加血管平滑肌细胞中腺苷酸环化酶的活性，并确定其在细胞因子诱导的诱导型一氧化氮合酶（iNOS）表达及核转录因子-κB（NF-κB）激活中的作用。此外，还研究了环磷酸腺苷（cAMP）和环磷酸鸟苷（cGMP）升高激动剂对IL-1β刺激的iNOS表达的相互作用。2. 将血管平滑肌细胞暴露于IL-1β会刺激cAMP的形成，但不会刺激cGMP的形成。细胞内cAMP水平在1小时内达到最大值，然后在接下来的5小时内逐渐下降。与异丙肾上腺素（在3×10⁻⁶ M时2分钟内约为9倍）引起的cAMP形成相比，这种IL-1β（60 U/ml）刺激的cAMP形成较为适度（在60 U/ml时1小时约为3倍）。3. 作为NO产生指标的亚硝酸盐积累，在IL-1β（60 U/ml，24小时）刺激下，可被cAMP依赖性磷酸二酯酶的选择性抑制剂（咯利普兰和曲喹辛）浓度依赖性地增加。这种效应可被cAMP依赖性蛋白激酶A的特异性激活剂Sp-8-CPT-cAMPS（10⁻⁴ M）重现，但可被cAMP依赖性蛋白激酶A的特异性抑制剂Rp-8-CPT-cAMPS（10⁻⁴ M）阻止。这些化合物单独使用[咯利普兰（10⁻⁶ M）、曲喹辛（3×10⁻⁶ M）和Sp-8-CPT-cAMPS（10⁻⁴ M）]会轻微但显著地增加一氧化氮的释放，而Rp-8-CPT-cAMPS则没有这种作用。4. 通过蛋白质印迹分析评估，在IL-1β（30 U/ml，24小时）刺激的平滑肌细胞中可诱导表达iNOS蛋白。在与咯利普兰（3×10⁻⁶ M）或Sp-8-CPT-cAMPS（10⁻⁴ M）联合暴露于IL-1β的平滑肌细胞中，iNOS蛋白水平显著增加，但在与Rp-8-CPT-cAMPS（10⁻⁴ M）联合暴露于IL-1β的细胞中则降低。在单独暴露于Sp-8-CPT-cAMPS或咯利普兰24小时的平滑肌细胞中发现iNOS蛋白有弱表达，而Rp-8-CPT-cAMPS没有这种作用。5. 通过电泳迁移率变动分析检测，将平滑肌细胞暴露于IL-1β（30 U/ml）30分钟会增加核提取物中NF-κB-DNA复合物的水平。在与Sp-8-CPT-cAMPS（10⁻⁴ M）、曲喹辛（10⁻⁶ M）或咯利普兰（10⁻⁶ M）联合暴露于IL-1β的细胞中发现类似水平的NF-κB-DNA复合物。这些调节剂单独使用均不影响NF-κB结合活性的基础水平。6. NO供体[硝普钠（SNP）10⁻⁴ M；二亚硝基铁-二-L-半胱氨酸复合物（DNIC），10⁻⁴ M；3-吗啉代西多胺（SIN-1），10⁻⁴ M]和心房利钠因子（10⁻⁶ M）显著增加了IL-1β（30或60 U/ml，24小时）刺激的iNOS蛋白表达和活性，这通过氧合血红蛋白转化为高铁血红蛋白间接评估。在没有IL-1β的情况下，SNP（10⁻⁴ M，24小时）但不是其他cGMP依赖性血管舒张剂会引起iNOS蛋白的适度表达。在与Rp-8-CPT-cAMPS（10⁻⁴ M）联合暴露于SNP的平滑肌细胞中未发现这种作用，而在与Sp-8-CPT-cAMPS（10⁻⁴ M）或咯利普兰（3×10⁻⁶ M）联合暴露于SNP的细胞中发现iNOS蛋白水平增加。7. 将血管平滑肌细胞暴露于S-亚硝基-L-半胱氨酸（Cys-SNO，10⁻⁴ M）、SNP（10⁻⁴ M）或SIN-1（10⁻⁴ M）35分钟对NF-κB的基础激活影响最小，但消除了由IL-1β（在最后30分钟添加30 U/ml）引起的激活。然而，在IL-1β刺激后（在30分钟暴露期的最后5分钟）添加Cys-SNO仅轻微降低了NF-κB-DNA复合物的水平。8. 这些数据表明cAMP依赖性途径起决定性作用
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