We now plan to further our hypothesis and determine the role of specific Aldo metabolites, present in kidney during the latent period, have on the expression and/or regulation of Aldo activity in kidney. We will focus on the physiological regulation of key specific hepatic and renal enzymes (5alpha- and 5beta-reductases, 3alpha- and 3beta-dehydrogenases, 2alpha-, 6alpha-, 6beta-, 16alpha-, 16beta-, 17alpha- P-450 hydroxylases) which generate active metabolites. In vivo and in vitro studies will be performed with male rat liver, kidney and specific segments of micro-dissected renal tubules using (3H)-Aldo and -Ring- A-reduced intermediates, and the results compared to (a) females, (b) males fed low or high Na+ diet, (c) males fed high K+ diet and (d) male rats following infusions of 3alpha, 5alpha- and 3beta, 5alpha-THAldo known to affect the magnitude of Na+ or K+ responses to Aldo. These same tissues will be used to determined which specific metabolites (e.g. 3beta, 5alpha- THAldo, carboxylic acid and sulfate derivatives) are activated by further transformation, following changes in dietary Na+ and K+, to metabolites which posses more MC activity. Together, these studies will indicate which metabolites are biologically important. We will complete the chemical identification (by NMR and LC-Mass Spec.) of each polar neutral hydroxylated metabolite of Aldo (NMA) synthesized, employing (17alpha)-stabilizing conditions, and test them biologically: (a) by acute or chronic administration for relative potency as mineralocorticoid (MC) agonists, and their ability to enhance or inhibit individual renal Na+ and K+ effects of Aldo; (b) chronically in intact rats following chronic infusion for feedback mechanisms on plasma renin activity and concentration, and plasma Aldo levels; (c) in rats fed either low Na+ of high K+ diet for possible enhancement of their MC potencies; (d) for their binding affinity for MC receptors and their effects on nuclear uptake of Aldo in kidney, and (e) their effects on Na+, K+-ATPase in kidney. Since liquorice derivatives cause glucocorticoids to express MC-like actions and enhance the Na+-retaining actions of aldo and DOC, glycyrrhetinic acid derivatives will be used as probes to determine (a) which common metabolic pathways are utilized by Aldo, Compd B and DOC and (b) their effects on the tissue uptake and receptor interactions of these three hormones. These proposed studies will determine the role of key metabolic pathways of Aldo (and DOC and corticosterone) in their mechanism of action as mineralocorticoids and in the pathogenesis of steroid-induced hypertension.
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