Induction of mRNA for tyrosine hydroxylase by cyclic AMP and glucocorticoids in a rat pheochromocytoma cell line: evidence for the regulation of tyrosine hydroxylase synthesis by multiple mechanisms in cells exposed to elevated levels of both inducing agents.

When rat pheochromocytoma PC18 cells are exposed to the cyclic AMP analog, 8-bromocyclic AMP, and/or the synthetic glucocorticoid, dexamethasone, there is a marked increase in the level of a single RNA species that hybridizes to the recombinant plasmid pTH.4, which contains sequences complementary to the RNA coding for tyrosine hydroxylase. This RNA species is 1800-1900 nucleotides in length and is presumably identical to an RNA species of similar size, isolated from rat pheochromocytoma PC8b cells and shown to code for tyrosine hydroxylase. Using RNA dot hybridization to quantitate the relative level of this tyrosine mRNA species, time course studies show that this mRNA increases relatively rapidly in PC18 cells treated with either 8-bromocyclic AMP or dexamethasone. A new steady state level of tyrosine hydroxylase mRNA is achieved after 6 hr or 12-24 hr of treatment with either 8-bromocyclic AMP or dexamethasone, respectively. The changes in the level of the mRNA slightly precede the changes in the rate of synthesis of tyrosine hydroxylase in cells treated with these inducing agents. After 24 hr of treatment with either 8-bromocyclic AMP or dexamethasone, the increases in the level of tyrosine hydroxylase mRNA are identical to the increases in the rate of synthesis of the enzyme in the cells. In cells treated simultaneously with both 8-bromocyclic AMP and dexamethasone, the increases in the enzyme level and rate of synthesis of tyrosine hydroxylase are approximately equal to the sum of the increases in these parameters observed in cells treated with either inducing agent alone. In contrast, there is not an additive increase in the level of tyrosine hydroxylase mRNA in cells treated with both inducing agents. This lack of an additive increase in mRNA for tyrosine hydroxylase is observed in total cellular RNA samples or in cytoplasmic RNA samples. Our results suggest that in cells exposed to elevated levels of either cyclic AMP or glucocorticoids, tyrosine hydroxylase is induced by a mechanism which increases the level of its mRNA, resulting in an increased rate of synthesis of the enzyme. However, in cells exposed to elevated levels of both cyclic AMP and dexamethasone, tyrosine hydroxylase enzyme levels are regulated by multiple mechanisms, one of which regulates the rate of synthesis of the enzyme without affecting the level of its mRNA.