INHIBITION OF PROTEIN-SYNTHESIS BY NITRIC-OXIDE CORRELATES WITH CYTOSTATIC ACTIVITY - NITRIC-OXIDE INDUCES PHOSPHORYLATION OF INITIATION-FACTOR EIF-2-ALPHA

Background: Nitric oxide (NO) is cytostatic for proliferating cells, i nhibits microbial growth, and down-regulates the synthesis of specific proteins. Studies were undertaken to determine the mechanism by which NO inhibits total protein synthesis and whether the inhibition correl ates with established cytostatic activities of NO. Materials and Metho ds: In in vitro experiments, various cell types were exposed to NO usi ng either donors or expression of inducible NO synthase (iNOS). The ca pacity of NO to suppress total protein synthesis, measured by incorpor ation of S-35-methionine into protein, was correlated with the capacit y of NO to suppress cell proliferation, viral replication, or iNOS exp ression. Phosphorylation of elF-2 alpha was examined as a possible mec hanism for the suppressed protein synthesis by NO. Results: Both NO do nors and expression of the iNOS suppressed total protein synthesis in L929 cells and A2008 human ovarian tumor cells in parallel with decrea sed cell proliferation. Suppressed protein synthesis was also shown to correlate with decreased vaccinia virus proliferation in murine perit oneal macrophages in an iNOS-dependent manner. Furthermore, iNOS expre ssion in pancreatic islets or RAW264.7 cells almost completely inhibit ed total protein synthesis, suggesting that nonspecific inhibition of protein synthesis may be the mechanism by which NO inhibited the synth esis of specific proteins such as insulin or iNOS itself. This possibi lity was confirmed in RAW264.7 cells where the in hibition of total pr otein synthesis correlated with the decreased iNOS protein. The decrea se in protein levels occurred without changes in iNOS mRNA levels, imp licating an inhibition of translation. Mechanistic studies revealed th at iNOS expression in RAW264.7 cells resulted in the phosphorylation o f eIF-2 alpha and inhibition of the 80S ribosomal complex formation. C onclusions: These results suggest that NO suppresses protein synthesis by stimulating the phosphorylation of eIF-2 alpha. Furthermore, our o bservations indicate that nonspecific inhibition of protein synthesis may be a generalized response of cells exposed to high levels of NO an d that inhibition of protein synthesis may contribute to many of the d escribed cytostatic actions of NO.