ROLE OF NITRIC OXIDE IN THE PATHOGENESIS OF LUNG DISEASE

人诱导型一氧化氮合酶(INOS)对一氧化氮的作用 一氧化氮(NO)的合成对炎症介质的反应。人类 INOS基因由26个外显子组成，编码131 kDa的蛋白质。本研究 旨在调查人类是否存在选择性剪接 Inos基因的表达。人肺泡巨噬细胞鼻腔和支气管的总RNA 上皮细胞和几个人体组织被转录成cDNAs和 应用特异引物聚合酶链式反应进行片段分析 Inos基因。四个选择性剪接位点被确定为 序列分析；这些包括：i)外显子5；ii)外显子8和 9；iii)外显子9、10和11；以及iv)外显子15和16。 新的iNOS cDNA的酸性序列预测了一个截短的蛋白质， 由外显子5缺失(149个碱基)引起，以及三个iNOS蛋白 基础结构删除。聚合酶链式反应产物的Southern分析结果一致 具有组织特异性的选择性剪接调节。在培养的A549中 (人肺泡II型上皮样肺癌细胞系)和 DLD1(一种人结肠腺癌)细胞，暴露于 细胞因子和脂多糖与诱导型一氧化氮合酶的增加有关 包括选择性剪接变异体的mRNA转录本。 由于iNOS作为二聚体是活跃的，因此选择性剪接的新形式 INOS可能参与了NO合成的调节。

Human inducible nitric oxide synthase (iNOS) is responsible for nitric oxide (NO) synthesis in response to inflammatory mediators. The human iNOS gene, containing 26 exons, encodes a protein of 131 kDa. This study was aimed at investigating the presence of alternative splicing of human iNOS mRNA. Total RNA from human alveolar macrophages, nasal and bronchial epithelial cells and several human tissues was transcribed to cDNA and analyzed using PCR with specific primers for segmental analysis of the iNOS gene. Four sites of alternative splicing were identified by sequence analysis; these included deletion of: i) exon 5; ii) exons 8 and 9; iii) exons 9, 10 and ll; and iv) exons 15 and 16. The deduced amino acid sequences of the novel iNOS cDNAs predict one truncated protein, resulting from exon 5 deletion (149bp), and three iNOS proteins with inframe deletions. Southern analyses of PCR products were consistent with tissue-specific regulation of alternative splicing. In cultured A549 (a human alveolar type II epithelium-like lung carcinoma cell line) and DLD1 (a human colorectal adenocarcinoma) cells, induction by exposure to cytokines and lipopolysaccharide was associated with an increase in iNOS mRNA transcripts including those of the alternatively spliced variants. Since iNOS is active as a dimer, the novel forms of alternatively spliced iNOS could be involved in regulation of NO synthesis.