The present proposal is designed to elucidate molecular mechanisms governing the gene expression and regulation of enzymes involved in catecholamine and acetylcholine biosynthesis. These enzymes are tyrosine hydroxylase, dopadecarboxylase, dopamine B-hydroxylase, and phenylethanolamine N-methyltransferase for catecholamine synthesis, and choline acetyltransferase for the biosynthesis of acetylcholine. Changes in neurotransmitter enzyme activity in the central and peripheral nervous systems and adrenal gland, caused by drugs, hormones, nervous injury and other factors will be studied by determining RNA concentrations and transcriptional rates as a means to further understand the mechanisms which regulate gene expression. Further, the regulation of phenotypic and gene expression during the embryonic development will be investigated. To carry out these investigations, the following strategy is employed: (a) clonal cell lines of neural and chromaffin cell origins will be used as simple systems to investigate the mechanisms governing enzyme induction, since they are easy to manipulate and will allow us to obtain large amounts of mRNA and nuclei; (b) using recombinant DNA technology, complementary DNA to enzyme RNA will be synthesized; (c) sensitive molecular hybridization procedures, such as Dot blot hybridization and in situ hybridization histochemistry using radiolabeled enzyme-cDNA probes, will be used to determine the quantity of specific enzyme mRNA; (d) in order to determine the rate of enzyme-mRNA synthesis, mRNA transcription will be measured in isolated nuclei of neuronal or chromaffin cells; and, finally, (e) these studies will be extended to the central and peripheral nervous systems and adrenal medulla. Thus, the present studies will provide, for the first time, a detailed insight into how alterations in the pharmacological, hormonal and physiological environment of neurons and chromaffin cells affected changes in gene expression of neurotransmitter synthesizing enzymes.
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