Tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, is expressed in adrenergic neurons and the adrenal medulla. We propose to study three systems in which TH is regulated by external factors. The first is glucocorticoid (dexamethasone) induction TH enzyme activity in clonal pheochromocytoma cell lines (PC12 and derivatives). This induction appears to be typical of steroid-induced transcription such that the steady state concentration of TH RNA is increased 3-10X. We proposed to determine a) if rate of TH transcription is increased (by assaying TH """"""""run-off"""""""" RNA in isolated nuclei); b) to identify flanking regulatory DNA sequences which bind the glucocorticoid receptor; c) to assay the biological function of such sequences by transfection of hormone-responsive transcription. The second project concerns how TH is induced in vivo by neuronal activity in sympathetic neurons and in the adrenal medulla. We plan to determine if increased trans-synaptic activity, mediated by the nicotinic acetylcholine receptor, increases TH transcription by measuring both steady state RNA levels and the rate of TH transcription. In addition we want to develop culture conditions in which trans-synaptic induction can be achieved in cell lines. Thirdly, we want to ask if the transmitter switch from adrenergic to cholinergic in primary sympathetic neurons, induced by non-neuronal cells or media from such cells, reflects changes in gene expression. Using TH probes, we will determine the nuclear and cytoplasmic TH RNA levels in adrenergic vs. cholinergic cultures. In addition, we want to clone the gene for choline acetyltransferase (CAT) the enzyme responsible for acetylcholine synthesis, since its activity is induced 50-1000X during the phenotypic switch. In addition, we propose to delineate the organization of the TH gene with regard to promoter, exon/intron and termination sites.
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