Short- and long-term effects of PACAP in PC12 cells: Phosphorylation and induction of tyrosine hydroxylase

332 SHORT- AND LONG-TERM EFFECTS OF PACAP IN PC12 CELLS: PHOSPHORYLATION AND INDUCTION OF TYROSINE HYDROXYLASE Randy Strong', Arun R. Wakade t, Akira Arimura # and John W. Haycock§ *GRECC, St. Louis VA Med Ctr, "Depts Pharmacology and Medicine, St. Louis Univ Sch Med, St. Louis, MO, tDept Pharmacology, Wayne State Univ Sch Med, Detroit, MI, *U.S./Japan Research Labs, Tulane Univ Sch Med, New Orleans, LA, §Dept Biochem Molec Biol, Louisiana State Univ Med Ctr, New Orleans, LA The peripheral, sympathoadrenal system exerts a major integrative function in homeostasis. Stressful extremes in the external or internal milieu produce increased sympathoadrenal activity, resulting in increased circulating levels and increased turnover of catecholamines. Secretion of catecholamines by the adrenal medulla is controlled by splanchnic nerve activity: At high rates of firing (e.g., 10 Hz), cholinergic stimulation of the adrenal medullary chromaffin cells predominates whereas the noncholinergic component prevails at lower, basal rates of firing (e.g., 1 Hz). Thus, the non-cholinergic component would be an appropriate signal for regulating quasi-steady state rates of catecholamine replenishment. The rate-limiting step in catecholamine biosynthesis is catalyzed by tyrosine hydroxylase (TH), and two physiological mechanisms for increasing biosynthetic rates are (1.) activation of existing TH molecules by phosphorylation and (2.) induction of the synthesis of TH itself resulting in more enzyme molecules. Numerous lines of evidence indicate that vasoactive intestinal polypeptide (VIP) is the non-cholinergic splanchnic nerve neurotransmitter in the rat. However, as developed in the preceding abstract by Wakade, et al., data that support a similar role for pituitary adenylate cyclase-activating polypeptide (PACAP) are also beginning to emerge. Thus, in the present studies, we compared the effects of VIP and PACAP on short- and long-term regulation of TH in a rat pheochromocytoma cell line (PC12). After preincubation with a2PO4, PC12 cells were treated with VIP or PACAP for 5 to 15 min. Both agents increased 32p incorporation into TH, however, PACAP was effective at 0.1-1 nM while 0.1-1 ~M VIP was required to produce an effect. The sitespecificity of TH phosphorylation produced by either agent was the same--VIP and PACAP both increased 32p incorporation into Ser31 and Ser4°. Phosphorylation of Ser4° is mediated by cAMP-dependent protein kinase whereas the phosphorylation of Ser31 is mediated by ERKs, a recently described family of MAP2 protein kinases activated by signalling pathways associated with increased diacylglycerol/inositol phosphate turnover. More prolonged treatment (6-24 h) of the PC12 cells with VIP or PACAP produced increases in the amount of TH protein per cell. Again, PACAP was 2-3 orders of magnitude more potent than VIP. Using Northern blots, an increase in TH mRNA transcripts was also observed, suggesting that both VIP and PACAP produce an increase in TH gene expression. Thus, both non-cholinergic neurotransmitter candidates appear to activate multiple signalling pathways involved both in signal transduction and gene expression related to the regulation of catecholamine function.