The intake of nicotine results in dramatic increases in the release and biosynthesis of catecholamines in the adrenal medulla, sympathetic ganglia and brain. The effects of nicotine on peripheral catecholaminergic systems are responsible for many of the pharmacological actions of the drug on the cardiovascular system, while its effects on brain catecholaminergic systems may participate in mediating some of the addictive properties of the drug. Catecholamine biosynthesis in both periphery and brain is controlled primarily by the activity of tyrosine hydroxylase. The studies outlined in this proposal test a number of hypotheses concerning the mechanisms by which nicotine regulates tyrosine hydroxylase gene expression in the periphery (the adrenal medulla) and in brain (locus coeruleus, substantia nigra and ventral tegmental area).
The specific aims are as follows: (1) to test the hypothesis that the effect of systemically-administered nicotine on TH gene expression in the adrenal medulla is partially due to transsynaptic mechanisms initiated in the brain; (2) to test whether nicotine induces TH and Th-mRNA in the brain by interaction with brain nicotinic receptors; (3) to test whether specific DNA response elements within the TH gene 5' flanking region mediate the stimulation of the gene in vivo after nicotine administration in transgenic mice; and (4) to identify members of the c-jun/c-fos and CREB/ATF family of transcription factors that are involved in the regulation of the Th gene in intact cells. Animals will be treated with nicotine and other drugs using different routes of administration and effects on TH activity, TH protein and TH-mRNA will be monitored in adrenal and brain. TH gene transcription rate will be measured in adrenal medulla. Transgenic mice that express a transgene containing wild type or mutagenized TH gene 5' flanking region fused to a reporter gene will be administered nicotine and the effects of the drug on reporter gene and endogenous TH gene expression will be monitored. PC12 cells will be used as a model system to test which transcription factors participate in the regulation of the TH gene in the intact cells. Antisense DNA, antisense RNA or dominant negative mutants will be used to """"""""knock-out"""""""" specific transcription factors in these experiments. These studies will increase our understanding of the mechanisms responsible for nicotine's effects on the cardiovascular system and will open up new avenues to study the mechanisms by which nicotine produces physical dependence, withdrawal and other aspects of addiction.
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