Hepatic and extrahepatic dehydrogenation/isomerization of 5-cholestene-3 beta,7 alpha-diol: localization of 3 beta-hydroxy-delta 5-C27-steroid dehydrogenase in pig tissues and subcellular fractions

Conversion of 5-cholestene-3 beta,7 alpha-diol (7 alpha-hydroxycholesterol) into 7 alpha-hydroxy-4-cholesten-3-one was studied with microsomes from different pig tissues and with liver subcellular fractions. Dehydrogenase/isomerase activity was efficient in microsomes from liver, ovary and lung, but less efficient in microsomes from adrenal gland and kidney. Microsomes from these tissues, with the exception of lung, were also active in dehydrogenation/isomerization of dehydroepiandrosterone and pregnenolone. Inhibition studies were carried out with trilostane, a competitive inhibitor of 3 beta-hydroxysteroid dehydrogenases active in steroid hormone biosynthesis (C19/C21-dehydrogenases), and a monoclonal antibody raised against a purified hepatic 3 beta-hydroxy-delta 5-C27-steroid dehydrogenase. The results showed that the C27-dehydrogenase activity in the tissues was not dependent on the C19/C21 dehydrogenases, but was dependent on the 3 beta-hydroxy-delta 5-C27-steroid dehydrogenase. Liver mitochondria, cytosol and peroxisomes lacked dehydrogenase/isomerase activity towards 7 alpha-hydroxycholesterol when microsomal contamination was taken into account. Immunoblotting experiments with monoclonal antibodies raised against the 3 beta-hydroxy-delta 5-C27-steroid dehydrogenase showed immunoreactivity only with protein in liver microsomes. Immunohistochemical studies showed localization of the 3 beta-hydroxy-delta 5-C27-steroid dehydrogenase in the bile duct epithelium. It is concluded that 7 alpha-hydroxycholesterol is converted into 7 alpha-hydroxy-4-cholesten-3-one by the microsomal 3 beta-hydroxy-delta 5-C27-steroid dehydrogenase in liver and extrahepatic tissues.     

Endogenous 7-oxocholesterol is an enzymatic product: characterization of 7 alpha-hydroxycholesterol dehydrogenase activity of hamster liver microsomes

Previously, we described a new metabolite derived from endogenous cholesterol in the presence of hamster liver microsomal protein and NADPH (Song et al., 1991, Biochem. Pharmacol. 41, 1439-1447). Through gas chromatography/mass spectral analysis of the metabolite and its methoxime-3-dimethyl-t-butylsilyl ether derivative, this metabolite has been definitively identified as 7-oxocholesterol. Isotope incorporation experiments using molecular 18O2 demonstrated that no oxygen atoms from molecular oxygen were incorporated into the product, 7-oxocholesterol, when 7 alpha-hydroxycholesterol was used as substrate. In contrast, one atom of 18O was incorporated into cholesterol from 18O2 during its metabolism to form 7 alpha-hydroxycholesterol. Formation of 7-oxocholesterol was dependent upon the presence of NADP+, 7 alpha-hydroxycholesterol, and hamster liver microsomes. This enzyme appears to be a membrane-bound protein and its activity was most abundant in liver microsomal fractions and to a lesser extent in mitochondrial fractions; little or no activity was observed in nuclei or cytosol. The enzyme activity was present in highest content in the livers of hamsters and was also observed in human and bovine liver microsomes, but not those of mouse, rabbit, or rat. The reaction was inhibited by 2'-AMP, but not by anti-NADPH:cytochrome-P450 oxidoreductase globulin, carbon monoxide, metyrapone, nor miconazole. In contrast to the previously characterized 3 beta-hydroxy-delta 5-C27-steroid oxidoreductase activity, NAD+ did not serve as an effective cofactor for 7-oxocholesterol formation. The ability of NADPH to partially serve as a cofactor in this reaction was shown to be due to a high NADPH-oxidase activity of hamster liver microsomes, thereby providing sufficient NADP+ to serve as the oxidizing pyridine nucleotide for the reaction. These results document the existence of a non-P450, NADP(+)-dependent 7 alpha-hydroxycholesterol dehydrogenase in liver microsomes which catalyzes this reaction. The product, 7-oxocholesterol, is produced enzymatically in the livers of hamsters and other mammals and may regulate bile acid metabolism or other processes due to its action as an oxysterol.     

11beta-hydroxysteroid dehydrogenase expression and activity in the human adrenal cortex

Although oxidation of cortisol or corticosterone by 11beta-hydroxysteroid dehydrogenase (11beta-HSD) represents the physiological mechanism conferring specificity for aldosterone on the mineralocorticoid receptor in mineralocorticoid target tissues, little attention has been paid until now to the expression and activity of this enzyme in human adrenals. We have shown that human adrenal cortex expresses 11beta-HSD type 2 (11beta-HSD2) gene, and found a marked 11beta-HSD2 activity in microsomal preparations obtained from slices of decapsulated normal human adrenal cortices. Under basal conditions, adrenal slices secreted, in addition to cortisol and corticosterone (B), sizeable amounts of cortisone and 11-dehydrocorticosterone (DH-B), the inactive forms to which the former glucocorticoids are converted by 11beta-HSD. Addition of the 11beta-HSD inhibitor glycyrrhetinic acid elicited a moderate rise in the production of cortisol and B and suppressed that of cortisone and DH-B. ACTH and angiotensin II evoked a marked rise in the secretion of cortisol and B, but unexpectedly depressed the release of cortisone and DH-B. ACTH also lowered the capacity of adrenal slices to convert [3H]cortisol to [3H]cortisone. This last effect of ACTH was concentration-dependently abolished by both aminoglutethimide and cyanoketone, which blocks early steps of steroid synthesis, but not by metyrapone, an inhibitor of 11beta-hydroxylase. Collectively, these findings indicate that the human adrenal cortex possesses an active 11beta-HSD2 engaged in the inactivation of newly formed glucocorticoids. The activity of this enzyme is negatively modulated by the main agonists of glucocorticoid secretion through an indirect mechanism, probably involving the rise in the intra-adrenal concentration of non-11beta-hydroxylated steroid hormones.     

N-glycosylation is not essential for enzyme activity of 11beta-hydroxysteroid dehydrogenase type 2

11Beta-hydroxysteroid dehydrogenase (11beta-HSD) catalyzes the oxidation of cortisol and corticosterone to cortisone and 11-dehydrocorticosterone, respectively. NAD-dependent 11beta-HSD is expressed at high levels in the distal nephron and contributes to mineralocorticoid specificity in that region. The present studies determined whether N-glycosylation is necessary for the activity of NAD-dependent 11beta-HSD (11beta-HSD2). First, cultured human colonic epithelial cells (T84 cells), which express native 11beta-HSD2 activity, were grown in medium with and without tunicamycin, an inhibitor of N-glycosylation. Tunicamycin had no effect on the enzyme activity. Next, the only putative N-glycosylation site (Asn394-Leu395-Ser396) of the cloned human kidney enzyme was eliminated by site-directed mutagenesis. Chinese hamster ovary (CHO) cells transfected with either the wild-type or the mutant cDNA construct showed no difference in the expressed enzyme activity, and Western blot analysis showed that the 11beta-HSD2 protein was the same size in cells expressing either the wild-type or the N394D mutant. Likewise, the molecular mass of the 11beta-HSD2 protein in T84 cells was not altered by treatment with peptide-N-glycosidase F or tunicamycin. We conclude that human 11beta-HSD2 is not a N-glycoprotein and N-glycosylation is not essential for the expression of enzyme activity.     

The uptake of calcium by isolated chromaffin granules of the adrenal medulla

11 beta-Hydroxysteroid dehydrogenase (11 beta-HSD) catalyses the interconversion of biologically active cortisol to inactive cortisone in man, and corticosterone to 11-dehydrocorticosterone in rodents. As such, this enzyme has been shown to confer aldosterone-selectivity on the mineralocorticoid receptor and to modulate cortisol/corticosterone access to the glucocorticoid receptor (GR). Two kinetically distinct isoforms of this enzyme have been characterized in both rodents and man; a low-affinity NADP(H)-dependent enzyme (11 beta-HSD1) which predominantly acts as an oxoreductase and, more recently, a high-affinity NAD-dependent uni-directional dehydrogenase (11 beta-HSD2). In this study we have analysed the expression of both 11 beta-HSD1 and 11 beta-HSD2 isoforms in rat adrenal cortex and medulla and have investigated their possible roles with respect to glucocorticoid-regulated enzymes mediating catecholamine biosynthesis in adrenal medullary chromaffin cells. Using a rat 11 beta-HSD1 probe and a recently cloned in-house mouse 11 beta-HSD2 cDNA probe, Northern blot analyses revealed expression of mRNA species encoding both 11 beta-HSD1 (1.4 kb) and 11 beta-HSD2 (1.9 kb) in the whole adrenal. Consistent with this, 11 beta-dehydrogenase activity (pmol 11-dehydrocorticosterone formed/mg protein per h, mean +/- S.E.M.) in adrenal homogenates, when incubated with 50 nM corticosterone in the presence of 200 microM NAD, was 97.0 +/- 9.0 and with 500 nM corticosterone in the presence of 200 microM NADP, was 98.0 +/- 1.4. 11-Oxoreductase activity (pmol corticosterone formed/mg protein per h) with 500 nM 11-dehydrocorticosterone in the presence of 200 microM NADPH, was 187.7 +/- 31.2. In situ hybridization studies of rat adrenal cortex and medulla using 35 S-labelled antisense 11 beta-HSD1 cRNA probe revealed specific localization of 11 beta-HSD1 mRNA expression predominantly to cells at the corticomedullary junction, most likely within the inner cortex. In contrast, 11 beta-HSD2 mRNA was more abundant in cortex versus medulla, and was more uniformly distributed over the adrenal gland. Negligible staining was detected using control sense probes. Ingestion of the 11 beta-HSD inhibitor, glycyrrhizic acid (> 100 mg/kg body weight per day for 4 days) resulted in significant inhibition of adrenal NADP-dependent (98.0 +/- 1.4 vs 42.5 +/- 0.4) and NAD-dependent (97.0 +/- 9.0 vs 73.2 +/- 6.7) 11 beta-dehydrogenase activity and 11-oxoreductase activity (187.7 +/- 31.2 vs 67.7 +/- 15.3). However, while levels of 11 beta-HSD1 mRNA were similarly reduced (0.85 +/- 0.07 vs 0.50 +/- 0.05 arbitrary units), those for 11 beta-HSD2 remained unchanged (0.44 +/- 0.03 vs 0.38 +/- 0.01). Levels of mRNA encoding the glucocorticoid-dependent enzyme phenylethanolamine N-methyltransferase which catalyses the conversion of noradrenaline to adrenaline, were also significantly reduced in those rats given glycyrrhizic acid (1.12 +/- 0.04 vs 0.78 +/- 0.04), while those for the glucocorticoid-independent enzyme tyrosine hydroxylase (1.9 kb), which catalyses the conversion of tyrosine to DOPA, were unchanged (0.64 +/- 0.04 vs 0.61 +/- 0.04). In conclusion, the rat adrenal gland expresses both 11 beta-HSD1 and 11 beta-HSD2 isoforms. 11 beta-HSD1 gene expression is localized to the adrenal cortico-medullary junction, where it is ideally placed to regulate the supply of cortex-derived corticosterone to the medullary chromaffin cells. This, together with our in vivo studies, suggests that 11 beta-HSD1 may play an important role with respect to adrenocorticosteroid regulation of adrenaline biosynthesis. The role of 11 beta-HSD2 in the adrenal remains to be elucidated.     

Activation of 11 beta-hydroxysteroid dehydrogenase by dehydroepiandrosterone sulphate as an anti-hypertensive agent in spontaneously hypertensive rats

The anti-hypertensive properties of dehydroepiandrosterone sulphate (DHEAS) have been investigated by studying its effects on blood pressure, on serum concentrations of corticosterone and dehydrocorticosterone, and on 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) activity in spontaneously hypertensive rats (SHR). SHR were given intraperitoneal injections of DHEAS (10 mg day-1 for 70 days) from six to 16 weeks of age. The blood pressure-time curve was significantly (P < 0.05) suppressed immediately after administration of DHEAS. There was no difference between the heart rates of control and DHEAS groups. Serum concentrations of corticosterone and dehydrocorticosterone in the DHEAS group were significantly (P < 0.05) lower than those of the control group. The dehydrocorticosterone/corticosterone concentration ratio was, however, significantly (P < 0.05) higher in the DHEAS group, suggesting that treatment with DHEAS enhanced the overall interconversion of corticosterone to dehydrocorticosterone. The activity of 11 beta-HSD in specific organs of the DHEAS group was affected, characteristic changes being increases in the kidney (14-58%), decreases in the liver (11-27%) and no change in the testis. Direct addition of DHEAS to 11 beta-HSD preparations from the kidneys of control SHR had the same effect as that observed in the in-vivo experiments. The fall in serum corticosterone in the DHEAS group is considered to be related, at least partly, to increased activity of kidney 11 beta-HSD. The inverse correlation of kidney 11 beta-HSD activity with serum corticosterone and blood pressure (-r = 0.628, P < 0.01, and -r = 0.478, P < 0.05, respectively) suggest that DHEAS delayed the development of hypertension in SHR by selective promotion of kidney 11 beta-HSD activity which in turn resulted in lower serum concentrations of corticosterone and its minimal aldosterone-like activity.     

11-beta-hydroxysteroid dehydrogenase activity and gene expression in the hypertensive Bianchi-Milan rat

OBJECTIVE: 11 beta-Hydroxysteroid dehydrogenase (11 beta-HSD), by converting the active steroids cortisol and corticosterone to their inactive metabolites, regulates steroid exposure to the mineralocorticoid and glucocorticoid receptors. We explored the hypothesis that a defect in 11 beta-HSD could result in overstimulation of either the mineralocorticoid or glucocorticoid receptors with subsequent hypertension in an established animal model of hypertension, the Bianchi-Milan hypertensive (BMH) rat. DESIGN AND METHODS: Groups of BMH rats with established hypertension (42-46 days old) and prehypertensive rats (22 days old) were compared with age-matched normotensive control rats. Kidney and liver 11 beta-HSD and glucocorticoid receptor messenger RNA (mRNA) levels were assessed by Northern and dot-blot analyses, and 11 beta-HSD activity as percentage conversion of [3H]-corticosterone to [3H]11-dehydrocorticosterone by tissue homogenate. RESULTS: Hepatic 11 beta-HSD activity and gene expression were significantly reduced in the hypertensive BMH rat compared with its normotensive genetic control. 11 beta-HSD activity was also reduced in the prehypertensive BMH rat (aged 25 days) from hypertensive parents, excluding hypertension per se as the cause of the abnormality. Plasma corticosterone was higher in the hypertensive rats. There was no difference in renal 11 beta-HSD activity or gene expression between hypertensive and normotensive BMH rats, or in glucocorticoid receptor gene expression in the liver or kidney. CONCLUSIONS: Normal levels of renal 11 beta-HSD mRNA and activity are found in the BMH rat. However, the hypertensive BMH rat does demonstrate impaired hepatic 11 beta-HSD activity which occurs at a pretranslational level, although it is not clear how this relates to the pathogenesis of hypertension in this model.     

Visual neglect in pretrigeminal cats with lateral suprasylvian lesions

11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) is a microsomal enzyme present in the peripheral tissues of the rat, including the liver, and is mediated by a number of factors in animal in vivo studies. However, the effect of peptide hormones and glucocorticoids on the activity of 11 beta-HSD in isolated rat hepatocytes is not clear. To investigate these effects, we determined 11 beta-HSD activity in a primary culture of rat hepatocytes by adding various concentrations of growth hormone, insulin and dexamethasone (Dex). 11 beta-HSD activity increased significantly after treatment with Dex (10(-9)M-10(-6)M) for 48h. Dex (100nM) treated hepatocytes, incubated for 12h to 48h, resulted in a significant two-to four-fold rise in 11 beta-HSD activity compared to control (p < 0.01), which was in contrast to GH (10(-9)M-10(-6)M) and insulin (10(-8)M-10(-5)M), which inhibited 11 beta-HSD activity (p < 0.05). These results suggest that the 11 beta-HSD of rat hepatocytes is under multifactorial regulation; Dex stimulates and GH and insulin inhibit 11 beta-HSD activity in primary cultures of rat hepatocytes.     

11Beta-hydroxysteroid dehydrogenase activity in progesterone biotransformation by the filamentous fungus Cochliobolus lunatus

Progesterone biotransformation was examined in relation to hydroxylating and dehydrogenating enzymes of Cochliobolus lunatus. 11beta-hydroxysteroid dehydrogenase activity (11beta-HSD) was located in cytosolic fraction and was NADP-dependent, inducible by progesterone and apparently uni-directional. Several inhibitors of 11beta-hydroxysteroid dehydrogenase were tested; furosemide, glycyrrhizic-acid and carbenoxolone did not influence the dehydrogenation of 11beta-hydroxy-4-pregnene-3,20-dione to 4-pregnene-3,11,20-trione, although grapefruit juice significantly reduced the rate of progesterone hydroxylation.     

11beta-hydroxysteroid dehydrogenase type 2 is the predominant isozyme in the guinea pig placenta: decreases in messenger ribonucleic acid and activity at term

The type 2 isozyme of 11beta-hydroxysteroid dehydrogenase (11beta-HSD2) is responsible for inactivating physiologically active glucocorticoids to their inert metabolites. This is the predominant 11beta-HSD isozyme in the human placenta, where it is believed to protect the fetus from high levels of maternal cortisol. Given the similarity in placental structure between the human and the guinea pig (hemomonochorial), we have evaluated the potential of utilizing the guinea pig as a model to study the function and regulation of placental 11beta-HSD2 in fetal development. In this study, we characterized the intrinsic properties of 11beta-HSD in the guinea pig placenta during late pregnancy. The 11beta-HSD activity in the placenta was characteristic of 11beta-HSD2 in that it possessed only dehydrogenase activity that was NAD-dependent and had a high affinity for cortisol (Km = 134 nM). Moreover, the level of the 11beta-HSD2-like activity decreased significantly at term. To verify the expression of 11beta-HSD2 gene and to determine whether corresponding changes in 11beta-HSD2 mRNA occur at term, we also cloned the cDNA encoding guinea pig placental 11beta-HSD2. The deduced guinea pig 11beta-HSD2 enzyme contains 395 amino acids and shares over 80% sequence identity with other mammalian 11beta-HSD2 proteins. Northern blot analyses demonstrated the presence of the mRNA for 11beta-HSD2 but not that for 11beta-HSD1. Moreover, the level of 11beta-HSD2 mRNA decreased significantly at term. The parallel decrease in levels of 11beta-HSD2 activity and mRNA at term is consistent with, and provides a plausible molecular basis for, the previously reported increase in the rate of placental transfer of cortisol between mother and fetus at that time. In conclusion, the present study demonstrates that the guinea pig resembles the human in that 11beta-HSD2 is the predominant, if not exclusive, isozyme expressed in the placenta. Therefore, the guinea pig appears to represent a suitable model in which to study the role of placental 11beta-HSD2 in human fetal development.     

Acute reduction of renal 11 beta-hydroxysteroid dehydrogenase activity by several antinatriuretic stimuli

The 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) activity of the kidney prevents access of cortisol or corticosterone to the renal mineralocorticoid receptor. Reduction of 11 beta-HSD activity by nutritional, hormonal, or pharmacologic factors might enhance the mineralocorticoid effect of these corticosteroids, thus causing sodium retention. To test this concept, we studied the effect on 11 beta-HSD activity of several antinatriuretic factors given orally to rats or exposed in vitro to rat renal tissue. Renal 11 beta-HSD activity was higher in fasted than fed rats (P < .05). Glucose, ethanol, and Toradol (Syntex Laboratories, Palo Alto, CA) given orally to fasted rats all reduced renal 11 beta-HSD activity by 20% to 40% (P < .05-.005) to levels similar to those observed in fed animals. Incubation of renal tissue from fasted rats with physiologic concentrations of insulin, ethanol, and Toradol also reduced 11 beta-HSD activity by 20% to 40% (P < .05-.01). These findings are consistent with the hypothesis that the antinatriuretic actions of these stimuli are due in part to alteration of renal 11 beta-HSD leading to greater mineralocorticoid effects in kidney.     

Structure and function of the hepatic form of 11 beta-hydroxysteroid dehydrogenase in the squirrel monkey, an animal model of glucocorticoid resistance

Both cortisol and aldosterone bind to and activate the mineralocorticoid receptor. Cortisol concentrations are generally 100- to 200-fold higher than aldosterone concentrations, yet mineralocorticoids clearly exert effects different from glucocorticoids. One hypothesis is that 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD), which converts cortisol to biologically inactive cortisone, protects the mineralocorticoid receptor from cortisol. The circulating concentrations of cortisol in the squirrel monkey are 20- to 50-fold higher than human cortisol concentrations, yet this animal has no evidence of glucocorticoid or mineralocorticoid excess. We used this experiment of nature to test the hypotheses that the known (hepatic) form of 11 beta-HSD protects renal mineralocorticoid receptors from the action of cortisol and that it modulates glucocorticoid concentrations in target tissues. Using a long oligonucleotide based on the rat sequence, we cloned the squirrel monkey 11 beta-HSD complementary DNA and gene. The encoded monkey amino acid sequence is 75% and 91% identical to the corresponding rat and human sequences, respectively. The tissue abundance of the messenger RNA for the monkey enzyme was similar to or less than that seen for the rat and human enzymes. Both the monkey and human 11 beta-HSD complementary DNAs were cloned into an expression vector and used to transfect cultures of Chinese hamster ovary cells. Both vectors were transcribed and translated into equivalent amounts of 11 beta-HSD enzyme. The monkey enzyme was slightly more efficient than the human enzyme in converting [3H]cortisol to cortisone, and estimates of the Michaelis-Menten constant and maximum velocity of both enzymes are similar. These data indicate that the abundance and activity of the hepatic form of 11 beta-HSD are insufficient to inactivate the very high concentrations of cortisol in the squirrel monkey, suggesting that this form of 11 beta-HSD does not defend the mineralocorticoid receptor or protect tissues from high cortisol concentrations. Rather, this enzyme appears to favor conversion of cortisone to cortisol, thus maximizing tissue concentrations of cortisol to overcome glucocorticoid resistance associated with a 50% reduction in glucococorticoid receptors.     

Distinct ontogeny of glucocorticoid and mineralocorticoid receptor and 11beta-hydroxysteroid dehydrogenase types I and II mRNAs in the fetal rat brain suggest a complex control of glucocorticoid actions

Glucocorticoids (GCs) act via intracellular mineralocorticoid (MR) and glucocorticoid receptors (GR). However, it has recently been recognized that GC access to receptors is determined by the presence of tissue-specific 11beta-hydroxysteroid dehydrogenases (11beta-HSDs) that catalyze the interconversion of active corticosterone and inert 11-dehydrocorticosterone. 11beta-HSD type 1 (11beta-HSD1) is a bidirectional enzyme in vitro that acts predominantly as a reductase (regenerating corticosterone) in intact neurons. In contrast, 11beta-HSD type 2 (11beta-HSD2) is a higher affinity exclusive dehydrogenase that excludes GCs from MR in the kidney, producing aldosterone-selectivity in vivo. We have examined the ontogeny of 11beta-HSD mRNAs and enzyme activity during prenatal brain development and correlated this with GR and MR mRNA development. These data reveal that (1) 11beta-HSD2 mRNA is highly expressed in all CNS regions during midgestation, but expression is dramatically reduced during the third trimester except in the thalamus and cerebellum; (2) 11beta-HSD2-like activity parallels closely the pattern of mRNA expression; (3) 11beta-HSD1 mRNA is absent from the CNS until the the third trimester, and activity is low or undectectable; and (4) GR mRNA is highly expressed throughout the brain from midgestation, but MR gene expression is absent until the last few days of gestation. High 11beta-HSD2 at midgestation may protect the developing brain from activation of GR by GCs. Late in gestation, repression of 11beta-HSD2 gene expression may allow increasing GC activation of GR and MR, permitting key GC-dependent neuronal and glial maturational events.     

Identification of a kinetically distinct activity of 11beta-hydroxysteroid dehydrogenase in rat Leydig cells

Leydig cells are susceptible to direct glucocorticoid-mediated inhibition of testosterone biosynthesis but can counteract the inhibition through 11beta-hydroxysteroid dehydrogenase (11beta-HSD), which oxidatively inactivates glucocorticoids. Of the two isoforms of 11beta-HSD that have been identified, type I is an NADP(H)-dependent oxidoreductase that is relatively insensitive to inhibition by end product and carbenoxolone (CBX). The type I form has been shown to be predominantly reductive in liver parenchymal cells and other tissues. In contrast, type II, which is postulated to confer specificity in mineralocorticoid receptor (MR)-mediated responses, acts as an NAD-dependent oxidase that is potently inhibited by both end product and CBX. The identity of the 11beta-HSD isoform in Leydig cells is uncertain, because the protein in this cell is recognized by an anti-type I 11beta-HSD antibody, but the activity is primarily oxidative, more closely resembling type II. The goal of the present study was to determine whether the kinetic properties of 11beta-HSD in Leydig cells are consistent with type I, type II, or neither. Leydig cells were purified from male Sprague-Dawley rats (250 g), and 11beta-HSD was evaluated in Leydig cells by measuring rates of oxidation and reduction, cofactor preference, and inhibition by end product and CBX. Leydig cells were assayed for type I and II 11beta-HSD and MR messenger RNAs (mRNAs), and for type I 11beta-HSD protein. Leydig cell 11beta-HSD had bidirectional catalytic activity that was NADP(H)-dependent. This is consistent with the hypothesis that type I 11beta-HSD is present in rat Leydig cells. However, unlike the type I 11beta-HSD in liver parenchymal cells, the Leydig cell 11beta-HSD was predominantly oxidative. Moreover, analysis of kinetics revealed two components, the first being low a Michaelis-Menten constant (Km) NADP-dependent oxidative activity with a Km of 41.5 +/- 9.3 nM and maximum velocity (Vmax) of 7.1 +/- 1.2 pmol x min x 10(6) cells. The second component consisted of high Km activities that were consistent with type I:NADP-dependent oxidative activity with Km of 5.87 +/- 0.46 microM and Vmax of 419 +/- 17 pmol x min x 10(6) cells, and NADPH-dependent reductive activity with Km of 0.892 +/- 0.051 microM and Vmax of 117 +/- 6 pmol x min x 10(6) cells. The results for end product and CBX inhibition were also inconsistent with a single kinetic activity in Leydig cells. Type I 11beta-HSD mRNA and protein were both present in Leydig cells, whereas type II mRNA was undetectable. We conclude that the low Km NADP-dependent oxidative activity of 11beta-HSD in Leydig cells does not confirm to the established characteristics of type I and may reside in a new form of this protein. We also demonstrated the presence of the mRNA for MR in Leydig cells, and the low Km component could allow for specificity in MR-mediated responses.     

Dehydrogenation of 3-phenoxybenzyl alcohol in isolated perfused rabbit skin, skin homogenate and purified dehydrogenases

The formation of 3-phenoxybenzoic acid from 3-phenoxybenzyl alcohol was determined in (a) rabbit ears, single-pass perfused with a protein-free buffer, pH 7.4; (b) the microsomal fraction and its supernatant from homogenized rabbit skin; and (c) purified alcohol dehydrogenase from horse liver and baker's yeast. The inhibition of product formation in (a) was about 60% by various 4-methylpyrazole concentrations, but metyrapone had no effect. Following ultracentrifugation, only the supernatant of homogenized skin showed product formation (apparent Vmay: 32 pmol/min per cm2 skin; apparent Km: 64 microM). 3-Phenoxybenzyl alcohol and ethanol dehydrogenation was similar by alcohol dehydrogenase from horse liver (apparent Km: 0.7 vs. 0.4 mM; apparent Vmax: 0.3 vs. 0.2 U/ microg protein). In baker's yeast, the apparent Km of 3-phenoxybenzoic acid formation was several times larger than that for ethanol dehydrogenation. The KI of 4-methylpyrazole for alcohol dehydrogenase from horse liver was 0.6 (3-phenoxybenzyl alcohol) vs. 0.04 microM (ethanol). The KI for ethanol in baker's yeast was 470 microM. In conclusion dehydrogenation is an important metabolic pathway in the skin for xenobiotics with an aliphatic alcohol at a side chain.     

11 beta-hydroxysteroid dehydrogenases of the choriocarcinoma cell line JEG-3 and their inhibition by glycyrrhetinic acid and other natural substances

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.     

Human hypertension caused by mutations in the 11 beta-hydroxysteroid dehydrogenase gene: a molecular analysis of apparent mineralocorticoid excess

CONVERSION OF CORTISOL TO CORTISONE: 11 beta-Hydroxysteroid dehydrogenase (11 beta-HSD) is a microsomal enzyme complex which, in humans, catalyses the interconversion between biologically active cortisol and inactive cortisone. This prereceptor signalling mechanism is essential for maintaining the aldosterone selectivity of the intrinsically non-specific mineralocorticoid receptor and for modulating glucocorticoid access to the glucocorticoid receptor. Apparent mineralocorticoid excess (AME) is a syndrome of severe low-renin mineralocorticoid hypertension associated with marked hypokalaemia which arises from a congenital deficiency of 11 beta-HSD. In AME patients, therefore, it is cortisol and not aldosterone which behaves as a potent mineralocorticoid. ISOFORMS OF 11 BETA-HSD: Two isoforms of human 11 beta-HSD have now been characterized and cloned. The type 1 isoform (11 beta-HSD1) is a low-affinity reduced nicotinamide adenine dinucleotide phosphate (NADP) dependent dehydrogenase-oxoreductase which is expressed in predominantly glucocorticoid target tissues and the encoding sequence of which is normal in patients with AME. In contrast, the type 2 isoform (11 beta-HSD2) is a high-affinity NADP-dependent unidirectional dehydrogenase which is expressed in placenta and mineralocorticoid target tissues such as renal collecting ducts and distal colonic epithelia. Exon- and intron-specific polymerase chain reaction amplification of the 11 beta-HSD2 gene from genomic DNA from members of a consanguinous kindred with AME consistently revealed a single missense mutation (C1228T) in two affected sibs and twin stillbirths. This mutation in codon 374 of exon 5 of the 11 beta-HSD2 gene creates an inframe premature stop (TGA) and, as such, results in a truncated 11 beta-HSD2 protein lacking the carboxyl-terminal proline-rich 32 amino acids. In keeping with an autosomal recessive mode of inheritance, both parents were phenotypically and biochemically normal but were heterozygous for this mutation. Unique to this kindred were expression analyses of the native mutant 11 beta-HSD2 enzyme in the stillbirth-affected placenta, which was almost completely devoid of NADP-dependent 11 beta-dehydrogenase activity. Immunohistochemical and Western blot analyses revealed the absence of 11 beta-HSD2 protein using antisera raised against synthetic peptide sequences corresponding either to the carboxyl terminus or other domains of the enzyme. MISSENSE MUTATION: In this kindred with AME, congenital deficiency of 11 beta-HSD activity is due to a single missense mutation in exon 5 of the 11 beta-HSD2 gene. Simultaneous studies by two other groups have similarly revealed no gross deletions or rearrangements of the 11 beta-HSD2 gene, but have described a number of single point mutations and oligonucleotide deletions in exons 3, 4 and 5, and adjacent to a splice site in intron 3. Recombinant expression analysis of site-directed mutant 11 beta-HSD2 complementary DNA constructs suggests a correlation between the predicted severity of these mutations and the biochemical and clinical phenotype. AME AS A CAUSE OF HYPERTENSION: The mutations in the 11 beta-HSD2 gene, together with those currently being sought by us for other kindreds with AME, establishes AME as a monogenic cause of human hypertension and will provide insight into the structure-function relationships of this important enzyme.     

Improved assay of hepatic microsomal cholesterol 7 alpha-hydroxylase activity by the use of hydroxypropyl-beta-cyclodextrin and an NADPH-regenerating system

Cholesterol 7 alpha-hydroxylase, the key enzyme in bile acid synthesis, has been implicated in atherosclerosis and gallstone disease. The aim of this study was to check if the use of hydroxypropyl-beta-cyclodextrin (HPBCD), a vehicle for solubilizing cholesterol, augmented the rate of 7 alpha-hydroxycholesterol formation in hamster liver microsomes compared to classical assays in which labeled cholesterol was delivered in Tween 80. We observed that [14C]cholesterol carried by HPBCD enhanced the sensitivity of the assay tenfold. However, linearity of 7 alpha-hydroxycholesterol formation with time was short because of the rapid transformation of 7 alpha-hydroxycholesterol into 7 alpha-hydroxy-cholesten-3-one and 7 alpha,12 alpha-dihydroxy-cholesten-3-one when NADPH alone was present in the incubation medium. In order to avoid the transformation of 7 alpha-hydroxycholesterol into 7 alpha-hydroxy-cholesten-3-one, which is essentially NAD(+)-dependent, but is also NADP(+)-dependent, NADPH (1 mmol/l) plus an NADPH-regenerating system must be present in the medium. In this improved assay, the optimal pH was 7.4 and the apparent Km for control and cholestyramine-fed hamsters had a similar value of 315 mumol/l; linearity in the formation of 7 alpha-hydroxycholesterol was also apparent after a relatively short time period (10 min), but with a markedly greater slope of the curve. With a short incubation time (6 min), microsomes from livers of hamsters (five and nine weeks old) that were fed with a commercial ground diet yielded rates of 7 alpha-hydroxycholesterol formation of 115 +/- 10 and 150 +/- 16 pmol/min.mg protein, respectively, whereas microsomes from hamsters fed with a lithogenic sucrose-rich diet (five weeks old) yielded rates of 7 alpha-hydroxycholesterol formation of 77 +/- 7 pmol/min.mg protein, which were significantly lower (-33%) than those of corresponding control hamsters. This improved cholesterol 7 alpha-hydroxylase assay is very sensitive, simple and rapid, and does not necessitate sophisticated equipment. It can be particularly useful for determining cholesterol 7 alpha-hydroxylase activity in liver biopsies from dyslipidemic or lithiasic patients.