N. Weiner

Treatment of a subclone of the PC12 pheochromocytoma cell line, PC8b, with either dexamethasone or 8-bromo cyclic AMP resulted in increased translational activity of tyrosine hydroxylase mRNA (mRNATH). Poly(A+)-containing RNA from cells treated with both inducers was used to construct a cDNA library. Double-stranded cDNA was inserted into the PstI site of pBR322 using GC tailing, and plasmids were used to transform Escherichia coli HB101. Colonies containing plasmids with inserted sequences were initially screened by DNA dot hybridization, and those positive colonies were then screened by hybrid selected translation. One plasmid, pTH.4, was identified as containing a 400-base pair sequence complementary to mRNATH. Nick-translated pTH.4 DNA was used to identify mRNATH as containing approximately 1800 nucleotides by Northern blot analysis. PC8b cells treated with either dexamethasone or 8-bromo cyclic AMP yielded greater mRNATH hybridization on Northern blot analysis and accumulated higher molecular weight tyrosine hydroxylase RNA species. Following treatment of cells with inducers, the temporal increase in tyrosine hydroxylase enzyme activity was associated in all cases with an increase in the translational activity and relative amount of mRNATH, and the fold increase in the latter two parameters was equal to or greater than the increase in enzyme activity.

In addition to its ability to release norepinephrine consequent to stimula­ tion, the adrenergic neuron can carry out a variety of functions related to the metabolism of its neurotransmitter. Both the adrenal medulla chromaffin cells (1-3) and the adrenergic neuron (3-5) store norepinephrine within vesicles which exhibit a characteristic dense core as seen in electron micro­ graphs which have been appropriately fixed prior to examination (6-14). Studies on isolated chromaffin granules and adrenergic vesicles have shown that the storage of catecholamines is associated with an active uptake pro­ cess from the adjacent cytoplasm (15-17). This uptake process appears to require A TP and Mg++ and is selectively inhibited by catecholamine deplet­ ing agents of the reserpine type (15, 16). In addition to the uptake process into isolated vesicles, the neuron concentrates norepinephrine across the ax­ onal membrane (18). This process is not inhibited by reserpine (19), but may be selectively blocked by other agents, among which are cocaine, imi­ pramine, and desmethylimipramine (20-23). The uptake of the catechol a­ mines and other phenylethylamines across the axonal membrane is a very rapid and efficient process. It is generally believed that this uptake is respon­ sible for the termination of the biological actions of either released or ad­ ministered catecholamines. Blockade of this uptake process leads to the po­ tentiation of the actions of norepinephrine and epinephrine (24-26). Norepinephrine is stored within chromaffin granules or adrenergic vesi­ cles largely in a bound form (27-30). The catecholamines appear to interact with ATP (31-34), resulting in the formation of a tetracatecholamine-ATP complex (35). This salt complex presumably is further bound to soluble proteins, the chromogranins, within the storage particle, although there is no direct evidence for the formation of a quantitatively significant complex (30, 36-38). Nevertheless, the ability of labelled catecholamines to enter the storage particles without exchanging with the bulk of the endogenous stores

Data demonstrating the direct phosphorylation of tyrosine hydroxylase [tyrosine 3-monooxygenase; L-tyrosine, tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2] purified from rat pheochromocytoma by ATP, Mg2+ and cyclic AMP-dependent protein kinase catalytic subunit are presented. The incorporation of phosphate is highly correlated with the activation of the hydroxylase when either the time of preincubation or the amount of protein kinase subunit is varied. The rate of phosphorylation of tyrosine hydroylase compares favorably with that of H1 histone, a known substrate of protein kinase. Lineweaver-Burk analysis of crude or purified rat pheochromocytoma tyrosine hydroxylase activity, as a function of pterin cofactor concentration, in the absence of ATP, Mg2+, and protein kinase catalytic subunit, yields a curvilinear relationship which can be resolved into two lines, suggesting two enzyme forms with different affinities for pterin cofactor. A fraction of the hydroxylase present in the tumor exists in the activated state, presumably due to the presence of ATP and endogenous protein kinase activity. When the solubl enzyme is activated by cyclic AMP, ATP, Mg2+, and protein kinase, virtually all of the enzyme is converted to the low Km state. We conclude that tyrosine hydroxylase is a substrate of cyclic AMP-dependent protein kinase in vitro and, presumably, in vivo.