Angiotensin II (AII) is the major regulator of aldosterone synthesis and has growth promoting actions. The revised application will utilize new genetic and molecular approaches to understanding the mechanisms and in vivo effects of the 12-Lipoxygenase (12-LO) pathway in mediating the actions of All. These approaches will utilize, a novel ribozyme or 'molecular scissors' approach to reduce 12-LO expression. Furthermore, a transgenic mouse overexpressing 12-LO and 12-LO 'knock-out' mouse will be utilized to test the in vivo role of 12-LO in mediating the steroidogenic and/or growth promoting actions of All in the adrenal. We will first evaluate the cellular and molecular mechanisms of 12-LO product action in the adrenal. A key aspect of these studies will be to investigate the role of specific forms of phosphatidic acid and activation of mitogen activated kinase (MAPK) cascades in mediating either the steroidogenic and/or growth promoting actions of All and LO pathway products. Exciting preliminary data suggests that All and the LO products can activate a new member of the MAPK family called cJun kinase (JNK) or stress activated kinase. Detailed studies will characterize the mechanisms of how All and the LO products activate (JNK) and the role of the specific forms of MAPK in mediating growth or steroidogenic response. These studies will utilize either pharmacologic inhibitors or dominant-negative mutant expression systems. A 12-LO ribozyme will be used to specifically block 12-LO activation in order to study the particular effects and signals for either growth or steroidogenesis. We will then determine the effects of changes in 12-Lo expression on adrenal glomerulosa steroidogenesis and growth. To more precisely evaluate the role of 12-LO and design human ribozymes, the human leucocyte type of 12-LO cDNA will be cloned. This cDNA will be overexpressed in human 295 cells, a cell line which in many respects resembles human glomerulosa cells. Nontransfected and transfected cells will be studied with respect to All-induced aldosterone synthesis and growth. We will determine the effects of low Na+ diet and All-fusion on glomerulosa histology and function in transgenic mice overexpressing 12-LO as well as 12-LO knock-out mice. These changes will be compared to effects induced by low Na+ diet and All infusion in the rat, which are well established models of All-induced adrenal growth and aldosterone synthesis. We will evaluate the role of 12-LO in excess aldosterone synthesis in a transgenic rat overexpressing renin in the adrenal. The completed studies should provide new mechanisms of All action on steroidogenesis and cell growth and provide the rationale for new molecular or pharmacologic approaches to reduce cardiovascular and renal disorders associated with enhanced All action.
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