Comparative study of induction of iNOS mRNA expression in vascular cells of different species.

Abstract

To determine the difference in induction of inducible nitric oxide synthase (iNOS) mRNA expression in cultured vascular cells of different species, the expression of iNOS genes and their regulatory mechanisms in rat, human, bovine, and rabbit vascular endothelial cells and smooth muscle cells (SMC) were studied by Northern blotting, chloramphenicol acetyltransferase (CAT) assay, and electrophoretic mobility shift assay (EMSA). Qualitative estimation of iNOS mRNA by Northern-blot analysis demonstrated that the combination of interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), and lipopolysaccharides (LPS) drastically induces iNOS expression in rat and human SMC, and a more moderate effect was observed for endothelial cells; the effect of IL-1 beta alone was much weaker than that of the three factors. IL-1 beta alone or a mixture of IL-1 beta, TNF-alpha, and LPS both showed negligible effect on iNOS expression in bovine and rabbit vascular endothelial cells and SMC. Results of CAT assay corresponded well with Northern analysis indicating 7-fold increase in CAT activity by the mixture of IL-1 beta, TNF-alpha, and LPS in SMC and more moderate, 2-fold increase, in endothelial cells. IL-1 beta alone produced an intermediate effect (less than 2-fold) on vascular SMC of rats and humans. The results of EMSA showed that two shifted bands appeared when the nuclear protein from rat and human vascular endothelial cells bound to the region from -1037 to -787 of the rat iNOS gene, while vascular SMC nuclear protein only produced a single shifted band under the same conditions. These results suggest that cell- and species-specific mechanisms exist in the induction of iNOS expression.