ROLE OF NITRIC OXIDE IN THE PATHOGENESIS OF LUNG DISEASE

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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.