We have now shown that purified extracts isolated from human urine inhibit, in a similar fashion to the licorice derivative glycyrrhetinic Acid (GA) the steroid inactivating enzymes 11beta-hydroxysteroid dehydrogenase (11beta-OHSD) and steroid 5beta-reductase (5beta-R) and may therefore confirm mineralocorticoid (MC)-like Na+ retaining activity on glucocorticoids (GCs). Since these endogenous substances mimic the inhibitory activities of Ga, we have named them """"""""Glycyrrhetinic Acid-Like factors"""""""" or GALFs, specifying them as 11beta- or 5beta-GALFs, respectively, their chemical nature is currently unknown. Having determined that urinary GALF levels ar markedly elevated in pregnancy, congestive heart failure and liver cirrhosis, all conditions of secondary hyperreninism, we now plan to focus our efforts on the large scale extraction of GALFs from urine of pregnant women. We plan to chemically characterize, following chromatographic purification and isolation, sufficient quantities of each GALF substance using GC- and LC- Mass Spectroscopy and high resolution NMR. We will also determine whether individual isolated GALF substances preferentially inhibit either 11beta-OHSD or 5beta-R activity and whether those GALFs present in pregnant urine possess similar chemical properties to those found in urine from normal males and females. At each step of purification, GALF fractions will be screened for their inhibitory activity against enzymatic preparations of: (a) renal and hepatic 11beta- OHSD (Isoform 1), (b) cortical collecting duct (CCD) cell and toad bladder 11beta-OHSD, (Isoform 2), (c) rat aorta 11beta-OHSD and (d) hepatic 5beta-R. The physiological relevance of each purified GALF substance will then be determined by measuring their ability in vivo to: (a) confer MC activity on the GC corticosterone, (b) amplify the Na+ retaining action of the MC's, Aldo and DOC, (c) regulate the expression of hepatic and renal 11beta-OHSD and 5beta-R at the transcriptional level, and in vitro (d) enhance Na+ transport produced by corticosterone in the isolated toad bladder. These studies are of fundamental importance since they will determine whether GALFs represent a new class of endogenous substances involved in the regulation of Na+ homeostasis and blood pressure.
