Regulation of type III nitric oxide synthase gene expression by nitric oxide

This study addressed the hypothesis that nitric oxide (NO) is able to regulate the expression of the Type III NO synthase (NOS) gene in bovine pulmonary endothelial cells (BPEC's). Monolayer cultures of BPEC's were transiently iransfected with a plasmid containing the complete 5' flanking region of the Type III NOS gene linked to the reporter gene luciferase. Following transfection, cells were incubated in the presence or absence of NO donors for 48 hours until harvesting for assay of luciferase activity. Both sodium nitroprusside (SNP) and 3-morpholinosydnonimine (SIN-1), at concentrations from 10^-7- 10^-4, caused a significant increase in reporter gene activity (SNP: 1.63-fold ±0.13, mean ±SD, n=3, p<0.05; SIN-1: 1.68-fold ±0.28, mean ± SD, n=3, p<0.05), indicating that NO upregulates the promoter activity of the Type III NOS gene. Preliminary studies have shown that a 6 hour exposure of BPEC's to 10^-5M SNP causes an increase in levels of Type III NOS mRNA and protein, determined by northern and western blot analysis. These results suggest that NO causes an increase in the level of Type HI NOS gene transcription in BPEC's, resulting in increased production of NOS mRNA and protein.