The long-term objectives of this research plan are to study and define the mechanisms of regulation of the normal arginine vasopressin (AVP) gene and the altered AVP gene in central diabetes insipidus. The regulation of AVP release will be evaluated under osmotic and non-osmotic stimulation. The specific hypothesis of the proposed research are as follows: 1. Specific structural features of the three domains of the AVP precursor (AVP, neurophysin, and glycopeptide) are necessary for the transport, processing and subsequent secretion of mature AVP. The protocols outlined in this proposal will define the region necessary for normal secretion of mature AVP from the cell and the processing defect with occurs in central diabetes insipidus in the Brattleboro rat. 2. Each regulatory element located in the promoter region of the AVP gene (CRE, GRE, AP2) has a specific role in the regulation of AVP gene expression. The described protocols will define the non-osmotic and osmotic regulation of this gene. The contribution of additional sites in the promoter region of the AVP gene to the regulation by these stimuli will also be assessed. The methods employed for these studies will include cell culture of COS cells and small cell lung cancer cells, mutagenesis, transfection, immunoprecipitation (pulse chase) immunofluorescence, measurement of AVP mRNA, and RIA of AVP. These studies should provide an understanding of the mechanisms of normal AVP secretion and the defect in hereditary central diabetes insipidus. Also the studies on the osmotic and non-osmotic controlling mechanisms of AVP gene expression will provide a greater understanding of AVP biosynthesis and release. The studies comprising this project are a logical molecular biological extension of this laboratory's long standing interest in the regulation of vasopressin release and action.
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