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The 11beta-hydroxysteroid dehydrogenase types 1 and 2 enzymes (11beta-HSD1 and 11beta-HSD2), modulate glucocorticoid occupation of the mineralocorticoid and glucocorticoid receptors by interconverting corticosterone and cortisol to the inactive metabolites 11-dehydrocorticosterone and cortisone within the target cells. The NAD(+)-dependent 11-HSD 2 in the kidney inactivates corticosterone and cortisol, allowing aldosterone, which is not metabolized, access to the receptor. Studies of the kinetics of 11-HSD 2 activity in the rat kidney have produced inconsistent results. Western blots done in the absence of the reducing agent beta-mercaptoethanol showed two bands with approximate MW of 40 and 80 kDa. When beta-mercaptoethanol was used, only the 40 kDa was detected, indicating that under non-denaturing conditions a significant proportion of the 11beta-HSD 2 exists as a dimer. NAD(+)-dependent conversion of 3H-corticosterone by 20 microg of microsomal protein increased approximately 10 fold with the addition of 5 mM DTT concentration. NADP(+)-dependent activity with 20 microg of microsomal protein was very low and did not change significantly when using DTT. In the presence of DTT, the predominant 11-HSD activity in the rat kidney is NAD(+)-dependent with a K(m) of 15.1 nM, similar to that of the cloned and expressed enzyme. These data suggest that dimerization and subsequent enzyme inactivation occur when protocols promoting oxidation of this protein are used.

The 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) enzymes convert corticosterone and cortisol to 11-dehydrocorticosterone and cortisone, and are thought to convey extrinsic specificity to the mineralocorticoid receptor by limiting access of the relatively more abundant glucocorticoids to it. Two different 11 beta-hydroxysteroid dehydrogenases (11 beta-HSD) have been described and cloned. The liver-type, NADP(+)-dependent 11 beta-HSD-1, has an affinity in the micromolar range and bidirectional activity. The NAD(+)-dependent 11 beta-HSD-2 has a higher affinity, in the nanomolar range, and exhibits only oxidase activity. 11 beta-HSD-2, because of its affinity and co-localization with the mineralocorticoid receptor, is likely to serve as the "gatekeeper" for the mineralocorticoid receptor in the kidney. Although the rat kidney expresses both isoforms, only the high-affinity, NAD(+)-dependent 11 beta-HSD-2 has been reported in the sheep kidney. We found both 11 beta-HSD NAD(+)- and NADP(+)-dependent activities in sheep kidney to be present. The NAD(+)-dependent activity exhibited a Km similar to that reported in the literature, 3.85 +/- 1.28 nM for corticosterone and 21.3 +/- 5.8 for cortisol, was distributed in approximately equal amounts between microsomes and nuclei, and was unidirectional, converting corticosterone to 11-dehydrocorticosterone. The enzyme exhibited prominent substrate inhibition. The NADP(+)-dependent activity had a Km for corticosterone of 4 +/- 1.3 nM for a Km for cortisol of 35.2 +/- 2 nM, 100-fold lower than that described for the 11 beta-HSD-1 in the liver of sheep and other species, and was more prevalent in the microsomes than the nuclei. This enzyme was not inhibited by its substrate. The NAD(+)-dependent activity was approximately 3-10 times greater than the NADP(+)-dependent activity when incubated with 5 nM corticosterone substrate, but had similar activity when incubated with 100 nM substrate concentrations. CHOP cells (a modified Chinese hamster ovary cell line) transiently transfected with the sheep 11 beta-HSD-2 plasmid exhibited a marked preference for NAD+ as co-factor. Oxidation of corticosterone by transfected cells in the presence of NADP+ was present, but minimal; NADP+ did not support the metabolism of cortisol, the primary glucocorticoid of sheep. These data suggest the existence of another NADP(+)-dependent enzyme, 11 beta-HSD-3, which, because of its high affinity and unidirectional oxidase activity, may play a physiological role in the modulation of glucocorticoid binding to both the mineralocorticoid and glucocorticoid receptors.

A method for the profiling of several important 3-oxo-4-ene urinary steroids is reported. The methodology is combined gas chromatography/mass spectrometry (GC/MS) utilizing stable isotope-labeled internal standards. The following standards were obtained or easily synthesized: [9, 11, 12, 12-2H4]cortisol, [1,2-2H2] and [9, 12, 12-2H2]cortisone, [1,2-2H2]6 beta-hydroxycortisol, and [1,2-2H2]18-hydroxycortisol. We found the following excretions of free steroids for normal adult males and females: cortisol (males mean +/- SD, 35 +/- 13; females mean +/- SD, 23 +/- 13), cortisone (males mean +/- SD, 58 +/- 23; females mean +/- SD, 50 +/- 22), 6 beta-hydroxycortisol (males mean +/- SD, 164 +/- 59; females mean +/- SD, 108 +/- 55), and 18-hydroxycortisol (males mean +/- SD, 148 +/- 55; females mean +/- SD, 71 +/- 30). For 18-hydroxycortisol in particular, the excretions were much higher for males than for females. We found that the larger part of urinary cortisol and cortisone is not free but is released from conjugation by enzymes present in snail digestive juice. Using a pooled urine sample from an equal number of male and female subjects, we found that for cortisol 29% was excreted free, 28% as glucuronide and 43% as other conjugates (probably sulfates). For cortisone 41% was free, 45% beta-glucuronide and 14% as other conjugates. Relatively little (3-8%) of the hydroxylated cortisols were excreted conjugated.

Essential hypertensive patients often respond to treatments mitigating mineralocorticoid action, even though circulating levels of these steroids are within normal ranges. In addition to the kidney, mineralocorticoid or Type I receptors are found in the brain and vascular smooth muscle where they mediate effects associated with several forms of experimental hypertension. Studies in which discrete anatomic or functional areas of the brain have been ablated demonstrate that the periventricular areas of the hypothalamus and the central sympathetic and baroreceptor systems are crucial for the development of hypertension in the renoprival, DOCA salt, and Dahl salt-sensitive rat. Intracerebroventricular (i.c.v.) infusion of aldosterone in both rats and dogs at doses that do not raise serum levels above normal produce hypertension. The hypertension produced by systemic mineralocorticoid excess, adrenal regeneration, and i.c.v. or oral administration of glycyrrhetinic acid or carbenoxolone in genetically normotensive rats and by dietary salt in the Dahl salt-sensitive rat is inhibited by the i.c.v. infusion of a mineralocorticoid receptor antagonist or a Na+ channel-selective amiloride analog. Recent data demonstrate the extraadrenal synthesis of steroids in aortic endothelial cells, smooth muscle cells and the brain. The role of the extraadrenal synthesis of steroids raises new avenues for research into the causes of hypertension.

Mineralocorticoid receptor (MR) selectivity for aldosterone is thought to be exerted by enzymes which inactivate competing glucocorticoids before they bind the receptor. Two different 11 beta-hydroxysteroid dehydrogenases (11 beta-HSD) have been described. 11 beta-HSD-1 is NADP(+)-dependent and has a Km in the micromolar range and bidirectional activity. 11 beta-HSD-2 is NAD(+)-dependent, has a Km in the nanomolar range, exhibits only oxidase activity, and colocalizes with the MR in the kidney, so is likely to serve as the gatekeeper for the MR. We have further characterized 11 beta-HSD activity in JEG-3 cells, a cell line derived from a human choriocarcinoma which was reported to have only the high affinity, NAD(+)-dependent 11 beta-HSD-2. We found that the Km for the conversion of corticosterone to 11-dehydrocorticosterone in intact cells and homogenates was about 16 nM. NAD(+)-dependent corticosterone conversion was equal in the nuclear and mitochondrial fractions and less, but significant, in the microsomal fraction. A high affinity, Km = 40 nM, NADP(+)-dependent enzyme was also found in homogenates. The subcellular distribution of this high affinity activity was greatest in the mitochondria, less in the nuclei, and even less, but still significant, in microsomes. Because of its cofactor dependency, high affinity, and different subcellular distribution, we suggest that this enzyme is neither the 11 beta-HSD-1 nor the 11 beta-HSD-2 and have named it 11 beta-HSD-3. Conversion of 11-dehydrocorticosterone to corticosterone did not occur in intact cells or in homogenates incubated with NADH or NADPH. Enzyme activity in intact cells was inhibited by glycyrrhetinic acid, carbenoxolone, progesterone, 5 beta-dihydroprogesterone, and 5 alpha-dihydroprogesterone, but not bile acids.

In this work we confirm by a metabolic method the existence of at least two enzymes with 11 beta- and 18-hydroxylase activities in rat adrenal mitochondria. The method was based on the ability of cortisol (F), a foreign alternative substrate, to inhibit competitively metabolite productions from various precursors. F inhibited a) aldosterone (ALDO) production from 11-deoxycorticosterone (DOC) without affecting the yields of corticosterone (B) and 18-hydroxy-11-deoxycorticosterone (18-OHDOC); b) 18-hydroxycorticosterone and aldosterone productions from B (Ki = 2.5 +/- 0.5 microM); and c) ALDO production from 18-OHDOC. These results suggest the existence of two categories of enzymes with both 11 beta- and 18-hydroxylase activities, one comprising those that catalyze the conversions of DOC to B and 18-OHDOC (F-insensitive reactions [FIS]) and the other one comprising the enzymes involved in the conversions of B to 18-OHB and ALDO and that of 18-OHDOC to ALDO (F-sensitive reactions [FS]). The cloned enzymes CYP11B1 and CYP11B2 would pertain respectively to the FIS and FS categories.

Aldosterone production from 11-deoxycorticosterone was stimulated by hemin in primary cultures and homogenates of calf adrenal zona glomerulosa, in a time- and dose-dependent fashion. The ferrochelatase inhibitor 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine (DDC) blocked the stimulation of aldosterone mediated by adrenocorticotropin (ACTH). Addition of hemin after treatment with DDC partially restored ACTH action. These results suggest that hemin may play a role in regulation of aldosterone production.

19-Nordeoxycorticosterone (19-norDOC) is a powerful mineralocorticoid that has been postulated to play a role in the pathogenesis of some forms of hypertension in the rat. We measured the daily excretion of 19-norDOC, aldosterone, and corticosterone in intact male and female SR/jr rats for 20 consecutive days. The excretion of corticosterone and aldosterone was higher during the first 4 days of collection and remained relatively stable for the rest of the collection period. The excretion of corticosterone and aldosterone was not different between male and female rats. The excretion of 19-norDOC, which, as has been reported previously, was significantly higher in female than male rats, varied over 600% from day to day in some individual rats. The variability in the excretion of 19-norDOC did not correlate with the excretion of aldosterone or corticosterone and did not appear to coincide with an estrous cycle. These studies also indicate that when the urinary excretion of steroids is intended to be used as an indication of steroid production in the basal state, a period of at least 4 days of acclimatization in metabolic cages, even for animals accustomed to handling, is necessary to obtain stable excretions.

We have prepared several monoclonal antibodies against aldosterone-3-carboxy-methyloxime-BSA conjugate by fusing spleen lymphocytes from an immunized mouse with the mouse myeloma line HL-1 Friendly. A total of 6 different clones were isolated and expanded. All of the antibodies exhibited low cross-reactivities against most of the compounds tested. Antibodies A5A3, A2E11, and C1E2 exhibited low cross-reactivity with 18-hydroxycorticosterone and 18-hydroxydeoxycorticosterone and showed no detectable displacement of tritiated aldosterone from the antibodies with cortisol, corticosterone, and related steroids. The only steroid that showed moderate cross-reactivity was 3 alpha,5 beta-tetrahydroaldosterone (around 3%). Clone A5H12 antibodies exhibited high cross-reactivity with tetrahydroaldosterone (19.3%) but otherwise was very similar to the above clones. Antibody of clone C1E4 showed high cross-reactivity to tetrahydroaldosterone (41.2%) and 18-hydroxyDOC (2%) with relatively low cross-reactivity to DOC (0.078%). Clone A2G9 antibodies were the only ones for which cortisol and corticosterone displaced tritiated aldosterone with cross-reactivities of 0.0042% and 0.125%, making them unsuitable for a direct radioimmunoassay of plasma aldosterone. The monoclonal antibodies were very sensitive to freezing and thawing. The cross-reactivities of the first three clones' antibodies compare favorably with those polyclonal antibodies that have been described to be suitable for use in direct radioimmunoassays of plasma aldosterone. Their advantage is the reliable supply of an antibody with consistent, predictable properties.

Mononephrectomized rats were given 1% NaCl solution to drink; half of them received 1 mg/day of 19-oxo-11 deoxycorticosterone acetate (19-oxo-DOCA) in sesame oil subcutaneously and half received only the oil for a period of four weeks. The steroid had no effect upon saline intake, systolic blood pressure, growth or the size of adrenals, hearts or kidneys, although it did produce hypernatremia and hypokalemia. The discrepancy between a demonstrable mineralocorticoid effect without blood pressure elevation awaits elucidation.

Four strains of mice were immunized with 6 different conjugates of 3-O (carboxymethyl-oximino)-18-hydroxycortisol to bovine serum albumin (3 preparations), turkey serum albumin, porcine thyroglobulin, and keyhole limpet hemocyanin. Spleens from 7 of 48 mice immunized were fused with Fox/NY and/or HL-1 Friendly myeloma cell lines, yielding many positive clones for antibody formation. Short cross-reactivities were done in 293 culture supernatants and were found to have low cross-reactivity (less than 0.001%) to cortisol, but very high cross-reactivity to 18-hydroxy-11-deoxycortisol (70 to 140%). One clone showed over 100% cross-reactivity with all the 18-hydroxylated steroids studied. The major problem encountered in the generation of monoclonal antibodies was the low antibody response in the vast majority of mice injected. Half the mice developed no measurable titer, and the clones evaluated from those that did produce antibodies cross-reacted with other 18-hydroxylated steroids. Nevertheless, the antibody developed could serve in radioimmunoassay for 18-hydroxy-11-deoxycortisol separated chromatographically from other cross-reacting steroids. This is important as no synthetic 18-hydroxy-11-deoxycortisol is available.