Multihormonal control of tyrosine aminotransferase in isolated liver cells.

The regulation of tyrosine aminotransferase activity by glucocorticoids and cyclic AMP was investigated in isolated liver parenchymal cell suspensions. The induction and maintenance of elevated levels of tyrosine aminotransferase activity in liver cells were completely dependent upon the presence of both the synthetic glucocorticoid, dexamethasone, and glucagon of dibutyryl cyclic AMP. No induction was observed when any of these compounds were tested alone. Immunotitration experiments revealed that the 6- to 7-fold increase in tyrosine aminotransferase activity following the addition of dexamethasone and glucagon was accompanied by a parallel increase in the amount of immunologically reactive enzyme protein. Pulse-labeling experiments established that this increase in enzyme protein could be fully accounted for by a corresponding increase in rate of synthesis of tyrosine aminotransferase. Neither hormone had any effect on the rate of degradation of the enzyme. The increase in tyrosine aminotransferase synthesis evoked by the presence of both hormones was blocked by the addition of actinomycin D or cycloheximide to the medium, demonstrating that RNA and protein synthesis were required for the induction of enzyme activity. By varying the time and order of addition of the inducers and inhibitions, evidence was obtained that the hormones act sequentially. The steroid hormone acts first, presumably to increase the level of functional tyrosine aminotransferase mRNA or its precursor. The processing of this precursor to a translatable form or the specific translation of tyrosine aminotransferase mRNA is apparently dependent upon a specific cyclic AMP-controlled process. In vivo experiments demonstrated that both glucocorticoids and cyclic AMP increase the level of functional tyrosine aminotransferase mRNA in the liver. The actions of the steroid hormone and cyclic nucleotide were blocked by alpha amanitin, establishing the requirement for ongoing gene transcription. The protein synthesis inhibitors, cycloheximide, emetine, and puromycin, were as effective as cyclic AMP in increasing tyrosine aminotransferase mRNA activity. The action of these inhibitors is probably related to their ability to elevate hepatic intracellular cyclic AMP levels, thus mimicking cyclic AMP administration. Extension of these in vivo studies to isolated liver cells will provide a valuable system for investigating the regulation of gene expression by glucocorticoids and cyclic AMP.