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

To determine the difference in induction of inducible nitric oxide synthase (iNOS) mRNA expression in cultured vascular cells of different species, th e expression of iNOS genes and their regulatory mechanisms in rat, human, b ovine, 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 estimat ion of iNOS mRNA by Northern-blot analysis demonstrated that the combinatio n 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 cel ls; the effect of IL- 1 beta alone was much weaker than that of the three f actors. 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 e ndothelial cells and SMC. Results of CAT assay corresponded well with North ern analysis indicating 7-fold increase in CAT activity by the mixture of I L-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 th an 2-fold) on vascular SMC of rats and humans. The results of EMSA showed t hat 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 ra t iNOS gene, while vascular SMC nuclear protein only produced a single shif ted band under the same conditions. These results suggest that cell- and sp ecies-specific mechanisms exist in the induction of iNOS expression.