Angiotensin II (AII) is the major regulator of aldosterone synthesis and is a potent vasoconstrictor. In addition, AII has potent growth promoting action in the adrenal and vascular smooth muscle. We have shown that the 12 and 15-lipoxygenase (LO) products, 12 and 15 hydroxyeicosatetraenoic acid (12 and 15-HETE) are key mediators of AII-induced steroidogenesis. In this renewal, we will evaluate several key mechanisms of LO product-induced aldosterone synthesis. Specifically, we will evaluate whether 12 and/or 15-LO products can increase aldosterone synthesis by activating the Ca2+-phospholipase protein kinase C (PKC) system. To specifically test whether AII and the LO products alter the expression of key aldosterone synthetic enzymes, we will quantitate the protein and mRNA levels respectively for side chain cleavage and specific isoforms of 11B, hydroxylase P450 enzymes. To investigate the role and mechanism of AII-induced adrenal growth, bovine cell line (AC1) which shows potent mitogenic actions to AII will be used. We will determine whether LO synthesis inhibition can alter AII-induced mitogenic actions. Similarly, the direct effect of LO product additions on adrenal cell growth will be determined in order to more specifically evaluate the effects and mechanism of growth promoting actions of 12 vs 15-LO products. In addition, the regulation of 12 and 15-LO enzyme activity and expression in the adrenal will be determined by studying whether Ca2+, diacylglycerol and PKC play a role in AII-induced LO synthesis. Finally, we will determine the cell specific localization of 12 and 15-LO protein and message in human adrenal tissue by performing immunohistochemistry and in situ hybridization with specific antisera and probes. We will also study the mechanism of AII-enhanced synthesis of 12 and 15-LO products. Specifically, the role of Ca2+, phospholipase C and D and PKC will be assessed. In addition, we will determine whether AII enhances the expression of 12 and 15-LO protein and mRNA levels. Our preliminary results are exciting and suggest that the LO products may enhance aldosterone synthesis by 1) increasing intracellular Ca2+, 2) activation of PKC and 3) inducing the expression of 11B, hydroxylase. Similarly, AII can enhance both 11B, hydroxylase and LO enzyme expression. These completed studies should provide key mechanisms of AII-induced aldosterone and growth promoting actions and therefore can provide the rationale for new therapeutic modalities to reduce cardiovascular and renal disorders associated with enhanced AII action.
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