The effect of endotoxin and glucocorticoid antagonizing factor (GAF) on the hormonal regulation of two hepatic enzymes, phosphoenolpyruvate carboxykinase (PEPCK) and tyrosine aminotransferase (TAT), is to be examined. The objective is to explain how GAF blocks the glucocorticoid induction of PEPCK but not that of TAT and why it has no effect on the induction of either enzyme by cAMP. Mice and hepatoma cells in culture will serve as models for this research. Three remaining sites where blockage of induction could arise are to be explored. They are transcription, post-transcriptional modification and translational events. The rate of change in enzyme mRNA synthesis during induction under control conditions and when endotoxin or GAF is present is to be evaluated. With GAF, both PEPCK and TAT will be studied since induction of the former but not the latter is inhibited. Enzyme mRNA formation will be measured by hybridization using the dot-blot and Northern transfer procedures and the results compared to those obtained by Northern transfer when nuclear mRNA is hybridized. This visualizes the processing of the primary transcript into mRNA. This will estimate total hybridizable mRNA present in the hepatoma cells or mouse liver. The methods can be used only with PEPCK since no cDNA probe is available for TAT. Translatable mRNA measured during in vitro protein synthesis will determine whether the change in translatable mRNA occurs in the same proportion as the change in hybridizable mRNA. One is by activity measurement and the other by radioimmunoassay. If the two change by equal amounts and agree with the mRNA changes, a reasonable estimate of the site of action of endotoxin or its mediator can be achieved (namely, transcription). If more mRNA appears by hybridization than by in vitro translation, it might be due to interference with normal post-transcriptional modification to active mRNA. Two dimensional gels will show how many cellular proteins fail to be induced by glucocorticoids when endotoxin is present. Purified GAF and antiserum to it will be used to assess the biological significance of GAF in endotoxemia. This research should advance insight into Gram-negative infections.
