Tyrosine hydroxylase (TH) protein and enzymatic activity are augmented with the long term activation of the sympathetic nervous system. This increase in TH activity is preceded by an elevation of TH mRNA which is known to result at least in part from an increase in transcription of the TH gene. However, recent evidence gathered in our laboratory as well as in others suggests that the more prolonged maintenance of increased TH mRNA may be the result, perhaps predominantly, of the enhanced stability of this transcript. The hypothesis of this proposal is that the regulation of the stability of TH mRNA is dependent on interactions between cis-acting elements associated with the 3' untranslated region (UTR) of this transcript and trans-acting proteins found in cytoplasmic extracts isolated from TH-releasing cells. The goals of this project are to [1] identify the regions of the TH 3' UTR which are involved in this process, [2] determine if activation of selected secondary messenger systems affect the magnitude and/or the site(s) of interaction of these trans-acting factors with the 3'UTR and [3] investigate the involvement of transcript stability in modulating the increase in TH mRNA which is associated with 2 different models of activation of the sympathetic nervous system, i.e., reserpine-induce hypotension and immobilization stress. The adrenal medulla will be utilized as a model of the sympathetic system. One of the unique features of the proposal is that a tetracycline (tet)-responsive expression system will be utilized which will place the control of expression of a hemagglutinin-tagged TH mRNA (hTHmRNA) under the exogenous control of this antibiotic. In the presence of tet transcription of hTHmRNA will be inhibited and the ensuing decline of this message will provide a direct index of transcript stability. RNA-protein mobility shift assays will be employed to study the molecular interactions between selected regions of the 3'UTR and cytoplasmic trans-acting proteins. Transgenic mouse modes will be utilized to study the in vivo role of the 3'UTR in the stress-related enhancement of adrenal medullary TH mRNA. The results obtained may lead to novel therapeutic approaches for regulating sympathetic nervous function via pharmacological manipulation of TH mRNA stability.
