Kaku Y, Nanri H, Sakimura T, Ejima K, Kuroiwa A, Ikeda M
Department of Health Development, University of Occupational and Environmental Health, Yahatanishi-ku, Kitakyushu, Japan.
Biochim Biophys Acta. 1997 Mar 27;1356(1):43-52. doi: 10.1016/s0167-4889(96)00156-5.
Exposure to various combinations of cytokines and lipopolysaccharide (LPS) has been reported to increase NO production in vascular endothelial cells. The molecular entity of the newly expressed nitric oxide synthase (NOS) in endothelial cells, however, has not yet been examined in detail. In this report, we carried out biochemical characterizations and molecular identification of NOS isoform(s) expressed in cytokine/LPS-treated bovine aortic endothelial cells (BAEC). The increased NOS activity in tumor necrosis factor-alpha (TNF-alpha)/LPS-treated BAEC was localized mainly in the cytosolic fraction and Ca2+-independent, whereas that in interferon-alpha,beta(IFN-alpha,beta)/LPS-treated BAEC was preferentially in the membrane fraction and Ca2+-dependent, suggesting that TNF-alpha/LPS increased an inducible NOS (iNOS)-like activity, and IFN-alpha,beta/LPS increased an endothelial constitutive NOS (ecNOS)-like activity. Correspondingly, the different responses to the cytokine/LPS pretreatment were demonstrated in semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) using primers specific for iNOS or ecNOS, that is, TNF-alpha/LPS elicited the expression of iNOS mRNA whereas IFN-alpha,beta/LPS increased that of ecNOS mRNA. A nuclear run-on transcription assay and an inhibition experiment by actinomycin D indicated that the apparent increase of ecNOS in the IFN-alpha,beta/LPS-treated BAEC was at least in part ascribed to the transcriptional activation. The nucleotide sequences of the amplified PCR products in TNF-alpha/LPS- and IFN-alpha,beta/LPS-treated BAEC were 93% and 99% identical to the corresponding regions of human hepatocyte iNOS and bovine ecNOS, respectively. These findings indicated that, in cytokine/LPS-treated BAEC, two NOS isoforms whose molecular natures were closely homologous to the conventional isoforms of iNOS and ecNOS were differently induced in response to distinct inflammatory stimuli.
据报道，暴露于细胞因子和脂多糖（LPS）的各种组合会增加血管内皮细胞中一氧化氮（NO）的产生。然而，内皮细胞中新表达的一氧化氮合酶（NOS）的分子实体尚未得到详细研究。在本报告中，我们对细胞因子/LPS处理的牛主动脉内皮细胞（BAEC）中表达的NOS亚型进行了生化特性分析和分子鉴定。肿瘤坏死因子-α（TNF-α）/LPS处理的BAEC中增加的NOS活性主要定位于胞质部分且不依赖于Ca2+，而干扰素-α、β（IFN-α、β）/LPS处理的BAEC中增加的NOS活性则优先定位于膜部分且依赖于Ca2+，这表明TNF-α/LPS增加了一种诱导型NOS（iNOS）样活性，而IFN-α、β/LPS增加了一种内皮型组成型NOS（ecNOS）样活性。相应地，使用针对iNOS或ecNOS的特异性引物进行的半定量逆转录-聚合酶链反应（RT-PCR）证明了对细胞因子/LPS预处理的不同反应，即TNF-α/LPS诱导iNOS mRNA的表达，而IFN-α、β/LPS增加ecNOS mRNA的表达。核转录延伸分析和放线菌素D抑制实验表明，IFN-α、β/LPS处理的BAEC中ecNOS的明显增加至少部分归因于转录激活。TNF-α/LPS和IFN-α、β/LPS处理的BAEC中扩增的PCR产物的核苷酸序列分别与人肝细胞iNOS和牛ecNOS的相应区域有93%和99%的同一性。这些发现表明，在细胞因子/LPS处理的BAEC中，两种分子性质与传统的iNOS和ecNOS亚型密切同源的NOS亚型在不同的炎症刺激下被不同地诱导。
