Phosphorylation and activation of tyrosine hydroxylase mediate the cAMP-induced increase in catecholamine biosynthesis in adrenal chromaffin cells.

Exogenous CAMP, dibutyryl CAMP, and 8-bromocAMP increased catecholamine biosynthesis from ~-[ l Cltyrosine in isolated purified bovine adrenal chromaffin cells. The acceleration of catecholamine biosynthesis occurred rapidly (within 2 min), was concentration dependent, persisted after the exogenous cAMP or cAMP analogue was removed, and dissipated after 30 min of incubation at 37 “C in the absence of nucleotide. Other cyclic nucleotides and adenine nucleotides (1 m ~ ) had no apparent effect upon catecholamine biosynthesis. 8-Bromo-CAMP did not influence 3,4-dihydroxyphenylalanine decarboxylation in situ, and neither exogenous pterin, exogenous epinephrine, nor elevated external tyrosine prevented the increase in catecholamine biosynthesis caused by 8-bromo-CAMP. Thus, the increase in tyrosine hydroxylation in situ appeared to result from an alteration in the catalytic properties of tyrosine hydroxylase per se. Tyrosine hydroxylase activity, in supernatants from hypotonically lysed cells, was elevated by prior treatment of the chromaffin cells with 8-bromo-CAMP. This activation dissipated (in parallel with biosynthesis rates) when previously treated cells were incubated at 37 “C in the absence of the nucleotide. In situ 8-bromoCAMP treatment and CAMP-dependent phosphorylating conditions in vitro both produced similar alterations in the kinetic constants for tyrosine and pterin cofactor. Further, the relative activation of tyrosine hydroxylase by cAMP in vitro was diminished by prior treatment of the intact cells with 8-bromo-CAMP. In supernatants prepared from lysed cells, endogenous CAMP-dependent protein kinase activity increased 32P incorporation (from [y3’P]ATP) into tyrosine hydroxylase. The concentration dependence of 32P incorporation in vitro was paralleled by tyrosine hydroxylase activity in vitro. More importantly, exogenous 8-bromo-CAMP increased 32P incorporation into tyrosine hydroxylase in intact chromaffin cells preincubated with 32Pi. These data support a direct relationship between CAMP-dependent phosphorylation and activation of the enzyme in vitro and demonstrate that similar processes can regulate catecholamine biosynthesis in situ. 14