Imai T, Hirata Y, Kanno K, Marumo F
Second Department of Internal Medicine, Tokyo Medical and Dental University, Japan.
J Clin Invest. 1994 Feb;93(2):543-9. doi: 10.1172/JCI117005.
By measurements of NO2-/NO3- (NOx) production and Northern blot analysis, we studied the effects of a membrane-permeable cAMP derivative, 8-bromo-cAMP, on the expression of inducible nitric oxide synthase (iNOS) gene and the synthesis of NOx in cultured rat vascular smooth muscle cells (VSMCs). 8-bromo-cAMP stimulated NOx production and increased steady-state levels of iNOS mRNA in rat VSMC in a time- and dose-dependent manner. NG-monomethyl-L-arginine, a NOS inhibitor, completely blocked the 8-bromo-cAMP-induced NOx production, whose effect was partially, but significantly reversed by an excess L-arginine, but not by D-arginine. Compounds that increase intracellular cAMP levels (cholera toxin, forskolin, and 3-isobutyl-1-methylxanthine), all stimulated NOx production. Dexamethasone inhibited the stimulated NOx production, as well as the induction of iNOS mRNA by cAMP. Both actinomycin D and cycloheximide completely blocked the stimulated NOx production by cAMP. Actinomycin D abolished the cAMP-induced iNOS mRNA, whereas cycloheximide remarkably increased iNOS mRNA levels in the presence and absence of 8-bromo-cAMP (superinduction). Actinomycin D, but not dexamethasone, completely abolished the cycloheximide-induced iNOS mRNA. The half-life of cAMP-induced iNOS mRNA was approximately 2 h, whereas no decay in the cycloheximide-induced iNOS mRNA was observed during 12 h. These results demonstrate that iNOS gene is upregulated by cAMP and the superinduction of iNOS mRNA is attributable to increased mRNA stability in rat VSMC.
通过测量NO2-/NO3-（NOx）的生成以及Northern印迹分析，我们研究了膜通透性cAMP衍生物8-溴-cAMP对培养的大鼠血管平滑肌细胞（VSMCs）中诱导型一氧化氮合酶（iNOS）基因表达和NOx合成的影响。8-溴-cAMP以时间和剂量依赖性方式刺激大鼠VSMC中NOx的生成并增加iNOS mRNA的稳态水平。NOS抑制剂NG-单甲基-L-精氨酸完全阻断了8-溴-cAMP诱导的NOx生成，过量的L-精氨酸可部分但显著逆转其作用，而D-精氨酸则无此作用。增加细胞内cAMP水平的化合物（霍乱毒素、福斯可林和3-异丁基-1-甲基黄嘌呤）均刺激了NOx的生成。地塞米松抑制了刺激的NOx生成以及cAMP对iNOS mRNA的诱导。放线菌素D和环己酰亚胺均完全阻断了cAMP刺激的NOx生成。放线菌素D消除了cAMP诱导的iNOS mRNA，而环己酰亚胺在存在和不存在8-溴-cAMP的情况下均显著增加了iNOS mRNA水平（超诱导）。放线菌素D而非地塞米松完全消除了环己酰亚胺诱导的iNOS mRNA。cAMP诱导的iNOS mRNA半衰期约为2小时，而在12小时内未观察到环己酰亚胺诱导的iNOS mRNA有衰减。这些结果表明，在大鼠VSMC中，iNOS基因被cAMP上调，且iNOS mRNA的超诱导归因于mRNA稳定性的增加。
