CELLULAR REGULATION OF TYROSINE HYDROXYLASE

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The long-term objectives of the research project are to describe the mechanisms by which catecholamine-containing neurons compensate for the diminution of endogenous catecholamine stores consequent to activity-related secretion of catecholamines. The compensatory replenishment of catecholamine stores results from an increase in catecholamine biosynthesis, the underlying basis of which is an increase in the catalytic activity of tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis. Stimulation-dependent phosphorylation of tyrosine hydroxylase is one mechanism that catecholaminergic cells employ to activate tyrosine hydroxylase. The specific objective of the proposed research is to determine the phosphorylation sites and the protein kinase system(s) involved in the phosphorylation of tyrosine hydroxylase in intact bovine adrenal chromaffin cells as stimulated by acetylcholine (the splanchnic nerve neurotransmitter). Limit tryptic digestion will be used to produce tyrosine hydroxylase phosphopeptides. After separation of the peptides, site-specific phosphorylation of tyrosine hydroxylase produced by acetylcholine in situ will be compared with site-specific phosphorylation of tyrosine hydroxylase produced in situ by selective activators of specific protein kinases and in vitro by various purified protein kinases. In addition, the effects of injecting purified protein kinases and protein kinase inhibitors directly into chromaffin cells will be evaluated for effects upon tyrosine hydroxylase phosphorylation and catecholamine biosynthesis. These data should allow identification of the protein kinase system(s) involved in regulating tyrosine hydroxylase phosphorylation in situ. Such studies should provide important insight into the regulation of catecholamine biosynthesis as well as a firm biochemical basis for the further study of the relationship between the phosphorylation and catalytic activity of tyrosine hydroxylase with reference specifically to its regulation in situ.