Increased risk of cardiovascular disease in tobacco users is in part due to chronic nicotine exposure. Nicotine in the peripheral blood stream is capable of stimulating the adrenal medulla to synthesize and release the catecholamine epinephrine, a potent neurohormone that elevates blood pressure and stimulates cardiac output. Prolonged elevated levels of epinephrine in the blood stream due to chronic nicotine exposure is associated with cardiovascular disease. The long-term objective of this research is to understand how the extracellular stimulus provided by nicotine mediates increased synthesis of the catecholamines in the adrenal medulla. Previous work has demonstrated that rats chronically exposed to nicotine have an increased activity and expression of the enzyme tyrosine hydroxylase (TH) in the adrenal medulla. TH catalyzes the rate limiting step in the biosynthetic pathway of the catecholamines. Understanding the mechanisms by which nicotine regulates the expression of the TH gene in the adrenal medulla may provide insight into the cardiac phathogenicity of cigarette smoking. Since adrenal chromaffin cells demonstrate increased levels of cellular cAMP in response to nicotine exposure, the specific aim of this project is to study the regulation of the rat TH gene promoter in response to cAMP. Using gel retardation assays and DNase I footprint assays potential regulatory elements of the rat TH gene promoter have been identified. By sequence comparison a number of these promoter elements contain sequences that share homology with known cAMP response elements (CREs). A reporter gene assay is presently being used to investigate the functional significance of these potential TH gene CREs. Mutant TH gene promoter constructs will be cloned into a vector upstream to the bacterial gene coding for chloramphenicol acetyl transferase (CAT). The mutated and wild type TH gene promoter CAT constructs will then be used for liposome mediated transient transfections in rat adrenal chromaffin tumor cell lines. The transfected cells in culture will be treated with cAMP analogs to examine the ability of response element mutations to block induction of CAT expression. Once functionally active cAMP inducible elements of the TH gene promoter have been identified the trans factor proteins interacting with these regions will be characterized. A modified southwestern blot technique will be used to determine the molecular weight of the DNA binding proteins. Ultimately, this same blotting technique will be used to clone the trans factor(s) binding to TH gene CREs by screening a rat adrenal expression library. These studies will provide significant insight into the biochemical events involved in chronic nicotine exposure and the signal transduction mechanisms that lead to increased expression of the TH gene.
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