No abstract

We have proposed that 11 beta-hydroxysteroid dehydrogenase is composed of structurally independent units with 11 beta-dehydrogenase and 11-reductase activities. We now report the purification of rat liver 11 beta-dehydrogenase to apparent homogeneity. Starting with microsomes, 800-fold purification was achieved with agarose-NADP affinity chromatography. No 11-reductase accompanied the purification. Homogeneity of 11 beta-dehydrogenase was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and amino acid end-group analysis and immunoprecipitation. The terminal amino acid was methionine. Monomer mol wt was 34,000. The enzyme was found to be a glycoprotein. A sequence of 40 amino acid units was identified from the amino end. The amino-terminal region was found to be highly nonpolar. Unlike unpurified microsomal 11 beta-dehydrogenase, which showed curvilinear Eadie plots, homogeneous enzyme gave rectilinear plots. Michaelis constants were 1.83 +/- 0.06 microM for corticosterone and 17.3 +/- 2.24 microM for cortisol. First order rate constants were 10 times greater for corticosterone than cortisol, and maximum velocities were similar.

In humans, glycyrrhetinic acid (GE), the active pharmacological ingredient of licorice, produces symptoms resembling those caused by excess mineralocorticoid secretion. We are proposing that 11 beta-dehydrogenase inhibition, and not intrinsic mineralocorticoid activity, is the primary mechanism of licorice induced pseudoaldosteronism. Glycyrrhizic acid (glycyrrhetinic acid glucuronide), when given orally to rats, partially inhibited renal 11 beta-dehydrogenase. In rats treated with dexamethasone before glycyrrhizic acid administration there was similar enzyme inhibition, suggesting that antimineralocorticoid effects of dexamethasone in licorice excess states are not mediated through a direct effect on 11 beta-dehydrogenase activity. Dispersed renal proximal tubular preparations, kidney homogenates, and microsomes readily converted corticosterone to 11-dehydrocorticosterone. GE and its synthetic analog carbenoxolone inhibited the conversion in these systems in a dose-dependent manner. Corticosteroid 11-oxoreductase, which was present in kidney homogenates at a level 10-20% that of 11 beta-dehydrogenase was not inhibited by any of the agents. With homogenate and microsomes, the Ki of GE was about 10(-9)-10(-8) M; with intact tubules, the Ki of GE was about 10(-5)-10(-6) M. It is suggested that a permeability barrier slows the entry of GE into the tubule cells. We conclude that the effects of licorice on corticosteroid metabolism in the kidney are based on its inhibition of 11 beta-dehydrogenase. Our data, supplemented by published evidence, is inconsistent with the conclusion that interaction with mineralocorticoid receptors accounts for the pharmacological effects of GE.

Corticosteroid 11 beta-dehydrogenase, the enzyme that catalyzes the oxidation of the biologically active steroid cortisol to its inactive metabolite cortisone, is present in testis. Since excess cortisol in men and other mammals and excess corticosterone in rodents cause physiological abnormalities including abnormal testicular function, it was pertinent to study the cellular distribution of 11 beta-dehydrogenase in the testis. Purified antiserum directed against homogeneous rat 11 beta-dehydrogenase was used to localize the enzyme in the developing rat testis. With immunofluorescence, the enzyme was not detectable in fetal testis or in the testis of young male rats until the 26th day of development. A few interstitial cells were stained in the testis of 26-day-old animals. In the testis of 31-day-old rats many cells in the interstitium were positive. In adult animals the entire interstitial region displayed bright fluorescence. Depleting animals of germ cells did not abolish the fluorescence. The appearance of this enzyme correlates temporally with the postnatal increase in Leydig cell number and the developmental rise in serum testosterone. We suggest that 11 beta-dehydrogenase of Leydig cells protects the testis from the deleterious effects of cortisol.