Shimizu T, Kinugawa K, Sugishita Y, Sugishita K, Harada K, Matsui H, Kohmoto O, Serizawa T, Takahashi T
Second Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Japan.
Cardiovasc Res. 1998 May;38(2):405-13. doi: 10.1016/s0008-6363(98)00005-4.
Inducible nitric oxide synthase (iNOS) has been implicated to contribute to myocardial dysfunction in various settings, but considerable species differences have been noted in the levels of iNOS expression and its function in several tissues. The aim of this study was to elucidate evolutional changes in myocardial iNOS expression and function.
An iNOS cDNA clone was isolated by RT-PCR from the 10-day old cultured chick embryonic ventricular myocytes stimulated with 10 micrograms/ml of lipopolysaccharide. Expression of the iNOS mRNA was analyzed with Northern blot analysis and RNase protection assay. The iNOS activity was estimated from conversion rates of L-arginine to L-citrulline and intracellular cGMP contents were measured with radioimmunoassay. Furthermore, both [Ca2+]i (fluorescent dye indo-1) and cell contraction (video motion detector) were simultaneously recorded.
Aside from the primer sequences, the insert (1026 bp) of the cDNA clone showed 66.4% identity at the deduced amino acid level to the human iNOS cDNAs. Northern blot analysis revealed that chicken iNOS mRNA of approximately 4.5 kb was induced by lipopolysaccharide within 6 h in the cultured myocytes. RNase protection assay also showed that lipopolysaccharide provoked 14.6 +/- 5.1-fold increases (n = 6, p < 0.05) in the iNOS mRNA signals within 6 h. The iNOS activity (+300%, P < 0.05) as well as the intracellular cGMP contents (+75%, P < 0.01) were significantly augmented in the lipopolysaccharide-stimulated cells. Both the cell contraction and [Ca2+]i were significantly reduced after the administration of a large amount (10 mM) of L-arginine in the myocytes pretreated with both lipopolysaccharide and NG-monomethyl-L-arginine (100 microM).
As like as the nucleotide and amino acid sequences, the myocardial effects of the iNOS may also be evolutionary conserved.
诱导型一氧化氮合酶（iNOS）在多种情况下被认为与心肌功能障碍有关，但在几种组织中，iNOS表达水平及其功能存在相当大的物种差异。本研究的目的是阐明心肌iNOS表达和功能的进化变化。
通过RT-PCR从用10微克/毫升脂多糖刺激的10日龄培养鸡胚心室肌细胞中分离出iNOS cDNA克隆。用Northern印迹分析和核糖核酸酶保护试验分析iNOS mRNA的表达。从L-精氨酸向L-瓜氨酸的转化率估计iNOS活性，并用放射免疫分析法测量细胞内cGMP含量。此外，同时记录[Ca2+]i（荧光染料indo-1）和细胞收缩（视频运动检测器）。
除引物序列外，cDNA克隆的插入片段（1026 bp）在推导氨基酸水平上与人类iNOS cDNA显示66.4%的同一性。Northern印迹分析显示，培养的心肌细胞中约4.5 kb的鸡iNOS mRNA在6小时内被脂多糖诱导。核糖核酸酶保护试验也显示，脂多糖在6小时内使iNOS mRNA信号增加了14.6±5.1倍（n = 6，p < 0.05）。脂多糖刺激的细胞中iNOS活性（+300%，P < 0.05）以及细胞内cGMP含量（+75%，P < 0.01）显著增加。在用脂多糖和NG-单甲基-L-精氨酸（100 microM）预处理的心肌细胞中，给予大量（10 mM）L-精氨酸后，细胞收缩和[Ca2+]i均显著降低。
与核苷酸和氨基酸序列一样，iNOS的心肌效应在进化上可能也是保守的。
