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PubMed2000

Distribution of glucocorticoid and mineralocorticoid receptors and 11beta-hydroxysteroid dehydrogenases in human and rat ocular tissues.

PURPOSE: The administration of glucocorticoids as topical or systemic medications may lead to the development of ocular hypertension through the induction of morphologic and biochemical changes in the trabecular meshwork leading to a reduction in the facility of aqueous outflow. Glucocorticoids exert their physiological effects by binding to and activating glucocorticoid and mineralocorticoid receptors. The activity of glucocorticoids is critically regulated at a prereceptor level by the two isozymes of 11beta-hydroxysteroid dehydrogenase. The purpose of this study was to determine the distribution of glucocorticoid target receptors and the isozymes of 11beta-hydroxysteroid dehydrogenase (11 beta-HSD) that regulate the activity of glucocorticoids at a prereceptor level in human and rat ocular tissues. METHODS: Horizontal sections of normal adult human and rat eyes were cut and hybridized with 35S-labeled cRNA probes specific for the glucocorticoid receptor, mineralocorticoid receptor, and 11beta-HSD types 1 and 2 using in situ hybridization. Immunohistochemical analysis of glucocorticoid and mineralocorticoid receptors using monoclonal antibodies was carried out on rat eye tissue sections. Whole rat eyes were homogenized and the activity of 11beta-HSD types 1 and 2 in the eye assessed as the percentage conversion of tritiated corticosterone to tritiated 11-dehydrocortico-sterone when corticosterone was added to the homogenate. RESULTS: In the rat ocular tissues mRNAs encoding glucocorticoid receptor, mineralocorticoid receptor, and 11beta-HSD types 1 and 2 were detected in nonpigmented ciliary epithelium, trabecular meshwork, corneal epithelium and endothelium, and anterior lens epithelium. Immunohistochemistry confirmed the presence of glucocorticoid and mineralocorticoid receptors at these sites. Activity of both isozymes of 11beta-HSD was demonstrated in homogenized rat eyes (percentage conversion of tritiated corticosterone to 11-dehydrocorticosterone; mean +/- SD, 11beta-HSD 1 = 15% +/- 5.3%, 11beta-HSD 2 = 7.9% +/- 2.8%). In both human and rat eyes, expression of mRNAs encoding glucocorticoid receptor and 11beta-HSD type 1 was high in the trabecular meshwork and lens epithelium, whereas expression of mRNAs encoding the mineralocorticoid receptor and 11beta-HSD type 2 was high in nonpigmented ciliary epithelium and corneal epithelium and endothelium. CONCLUSIONS: Glucocorticoid target receptors and the enzymes regulating glucocorticoid activity at these receptors are present in mammalian ocular tissues, which regulate aqueous humor formation and outflow. Alteration in the number or affinity of receptors or in the activity of regulatory enzymes may alter the susceptibility of certain individuals to the effects of glucocorticoids on intraocular pressure.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1991

Effect of estradiol on spermatogenesis and testicular hydroxysteroid dehydrogenase activities in Bidder's organectomized toad (Bufo melanostictus).

Inhibition of spermatogenic activity and increase in Leydig Cell nuclear area (L C N A), testicular delta 5-3 beta-hydroxysteroid dehydrogenase (delta 5-3 beta-HSD) and 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) activities were noted after Bidder's organectomy. Administration of estradiol in Bidder's organectomized toad showed more or less similar result as the control animal. It is supposed that estradiol coming from the Bidder's organ might play a role in normal testicular activities.

17-Hydroxysteroid Dehydrogenases↗
PubMed1990

Direct effects of lithium chloride on testicular delta 5-3 beta and 17 beta-hydroxysteroid dehydrogenase activities in the rat--in vitro study.

Testicular delta 5-3 beta- and 17 beta-hydroxysteroid dehydrogenase (delta 5-3 beta- and 17 beta-HSD) activities of rat are inhibited in vitro by a wide range of lithium concentration. The inhibitory effects of lithium are evident at a concentration of 2.5 mM which is easily achieved during the treatment of acute manic patients with lithium. This suggests that lithium exerts a direct inhibitory effect on testicular hydroxysteroid dehydrogenase activities.

17-Hydroxysteroid Dehydrogenases↗
PubMed1985

Purification and properties of 3 alpha-hydroxysteroid dehydrogenase from rat brain cytosol. Inhibition by nonsteroidal anti-inflammatory drugs and progestins.

The 3 alpha-hydroxysteroid dehydrogenase (EC 1.1.1.50) of rat brain cytosol has been purified to apparent homogeneity. The purification procedure involves six successive steps, includes one affinity chromatography, and yields enzyme which displays a 1,550-fold enhancement in specific activity. The homogeneous enzyme has a Km of 8.0 microM for 5 alpha-dihydrotestosterone, a Vmax of 1.3 mumol of 3 alpha-androstanediol formed per h/mg of protein, and displays a preference for NADPH. It appears to be the major activity responsible for the reduction of 5 alpha-dihydrotestosterone in this tissue and may play a pivotal role in brain androgen metabolism. The homogeneous enzyme has several properties in common with the 3 alpha-hydroxysteroid dehydrogenase purified from rat liver cytosol (Penning, T. M., Mukharji, I., Barrows, S., and Talalay, P. (1984) Biochem. J. 222, 601-611). It is a monomer with a molecular weight of 31,000, it has a pI of 5.5, and it is potently inhibited by the nonsteroidal anti-inflammatory drugs (IC50 value for indomethacin = 2.0 microM). The potency of inhibition observed for the brain enzyme parallels that observed for cyclooxygenase: indomethacin greater than fenamates greater than l-methylpyrrole acetic acids greater than arylpropionic acids greater than salicylates greater than acetaminophen. Examination of a variety of steroidal contraceptives as modulators of the dehydrogenase indicates that ethinylestradiol is a very poor inhibitor (IC50 = 100 microM), while 6-medroxyprogesterone acetate (Provera) is an extremely potent inhibitor (IC50 = 0.2 microM). The possibility exists that brain androgen metabolism may be altered by the nonsteroidal anti-inflammatory drugs and synthetic progestins.

3-Hydroxysteroid Dehydrogenases↗
PubMed1983

Inactivation of human placental 17 beta,20 alpha-hydroxysteroid dehydrogenase by 16-methylene estrone, an affinity alkylator enzymatically generated from 16-methylene estradiol-17 beta.

The substrate 16-methylene estra-1,3,5(10)-triene-3,17 beta-diol (16-methylene estradiol-17 beta) and its enzyme-generated alkylating product, 3-hydroxy-16-methylene estra-1,3,5(10)-triene-17-one (16-methylene estrone), were synthesized to study the 17 beta- and 20 alpha-hydroxysteroid dehydrogenase activities which coexist in homogeneous enzyme purified from human placental cytosol. 16-Methylene estradiol, an excellent substrate (Km = 8.0 microM; Vmax = 2.8 mumol/mg/min) when enzymatically oxidized to 16-methylene estrone in the presence of NAD+ (256 microM), inactivates simultaneously the 17 beta- and 20 alpha-activities in a time-dependent and irreversible manner following pseudo-first order kinetics (t1/2 = 1.0 h, 100 microM, pH 9.2). 16-Methylene estradiol does not inactivate the enzyme in the absence of NAD+. 16-Methylene estrone (Km = 2.7 microM; Vmax = 2.9 mumol/mg/min) is an affinity alkylator (biomolecular rate constant k'3 = 63.3 liters/mol-s, pH 9.2; KI = 261 microM; k3 = 8.0 X 10(-4) S-1, pH 7.0) which also simultaneously inhibits both activities in an irreversible time-dependent manner (at 25 microM; t1/2 = 7.2 min, pH 9.2; t1/2 = 2.7 h, pH 7.0). Substrates (estradiol-17 beta, estrone, and progesterone) protect against inhibition of enzyme activity by 16-methylene estrone and 16-methylene estradiol. Affinity radioalkylation studies using 16-methylene [6,7-3H]estrone demonstrate that 1 mol of alkylator binds per mol of inactivated enzyme dimer. Thus, 16-methylene estradiol functions as a unique substrate for the enzymatic generation of a powerful affinity alkylator of 17 beta,20 alpha-hydroxysteroid dehydrogenase and should be a useful pharmacological tool.

20-Hydroxysteroid Dehydrogenases↗
PubMed1981

Enzymatic determination of bile acids from liver cells with 3 alpha-hydroxysteroid dehydrogenase--a warning.

3 alpha-Hydroxysteroid dehydrogenase was used to monitor bile acid production by rat hepatocyte suspensions. Several observations suggested that the method lacked specificity and detected some other metabolites besides bile acids. This was due to a malate dehydrogenase activity present in four different commercial enzyme preparations of 3 alpha-hydroxysteroid dehydrogenase. It is concluded that considerable error can be caused by this contaminating malate dehydrogenase activity, especially in the case of low bile acid concentration in the sample.

3-Hydroxysteroid Dehydrogenases↗
PubMed1994

NAD(+)-dependent isoform of 11 beta-hydroxysteroid dehydrogenase. Cloning and characterization of cDNA from sheep kidney.

11 beta-Hydroxysteroid dehydrogenase (11-HSD) catalyzes the conversion of cortisol to cortisone and corticosterone to 11-dehydrocorticosterone. This activity may be required to confer normal ligand specificity upon the mineralocorticoid receptor. Although an isozyme of 11-HSD was previously isolated from rat liver, a different isozyme is apparently expressed in mineralocorticoid target tissues. We isolated a sheep kidney cDNA clone encoding this isozyme by expression screening using Xenopus oocytes. The cDNA is 1.8 kilobase pairs in length and encodes a protein of 427 amino acid residues with a predicted M(r) of 46,700. When expressed in oocytes, this enzyme functions as an NAD(+)-dependent 11 beta-dehydrogenase with very high affinity for steroids, but it has no detectable reductase activity. It is 37% identical in amino acid sequence to an NAD(+)-dependent isozyme of 17 beta-hydroxysteroid dehydrogenase but only 20% identical to the NADP(+)-dependent liver isozyme of 11-HSD. It is expressed at high levels in the kidney and adrenal and at lower levels in the colon. The corresponding gene is present in a single copy in the sheep genome. In humans, this gene is a candidate locus for the syndrome of apparent mineralocorticoid excess, a form of hypertension postulated to result from 11-HSD deficiency in mineralocorticoid target tissues.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1993

Glucocorticoid metabolism in the cardiac interstitium: 11 beta-hydroxysteroid dehydrogenase activity in cardiac fibroblasts.

The interstitial fibrosis seen in the heart and systemic organs in states of primary or secondary mineralocorticoid excess suggests that fibroblasts are responsive to mineralocorticoid. In vitro studies demonstrating increased fibroblast collagen synthesis in response to MC are consonant with this view. The nicotinamide-adenine dinucleotide phosphate(+)-dependent enzyme 11 beta-hydroxysteroid dehydrogenase converts the glucocorticoids corticosterone and cortisol to the inactive metabolites 11-dehydrocorticosterone and cortisone, respectively, conferring mineralocorticoid specificity to the cells within which it is active. We investigated the presence of 11 beta-hydroxysteroid dehydrogenase in sonicates of cultured vascular endothelial cells and cardiac fibroblasts by incubating sonicates for 1 hour in the presence of 5 x 10(-9) mol/L tritiated corticosterone or tritiated cortisol (1 microCi) and using reverse-phase high-performance liquid chromatography coupled to an on-line radioisotope detector for steroid separation and quantitation. Extracts of bovine endothelial cells showed no enzymatic activity with either substrate, whereas extracts of rat cardiac fibroblasts readily converted corticosterone to 11-dehydrocorticosterone, even in the absence of exogenous nicotinamide-adenine dinucleotide phosphate+ (10% conversion). When 5 x 10(-4) mol/L nicotinamide-adenine dinucleotide phosphate+ was added to sonicated fibroblasts, conversion increased to 50%, corresponding to 12 pmol 11-dehydrocorticosterone formed/mg protein. Conversion of cortisol to cortisone was not observed in fibroblast or endothelial cell extracts. Significant levels of corticosterone to 11-dehydrocorticosterone conversion (0.14 pmol/10(6) cells/hour) were detected in intact fibroblasts, but no 11-dehydrogenation of corticosterone was observed in intact endothelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)

11-Hydroxycorticosteroids↗
PubMed1979

Immobilized hydroxysteroid dehydrogenases for the transformation of steroids in water--organic solvent systems.

The hydroxysteroid dehydrogenases: beta-HSDH, 20 beta-HSDH, and 3 alpha-HSDH, were immobilized on CNBr-activated Sepharose. The effect of various immobilization conditions on the activity recovery and stability were examined. The presence of cofactor during the immobilization reaction increased the activity recovery (40--60% of the total) and also led to materials highly stable in the presence of organic solvents. For example, beta-HSDH maintained 60% of its original activity two months after continuous use in the water--ethyl-acetate system. Kinetic experiments showed that the increase of the apparent Km values is poor and demonstrated that the organic solvent behaves as a weak inhibitor (ki greater than 0.2M) for the substrate. The immobilized enzymes lyophilized in the presence of sucrose had full activity restored even after several months storage at room temperature. Immobilized hydroxysteroid dehydrogenases were shown to be suitable for preparative transformation of steroids in water--organic solvent systems.

Cortisone↗
PubMed1976

Mass spectra of delta 4- and 5 alpha-3-ketosteroids formed during the oxidation of some 3 beta-hydroxysteroids by cholesterol oxidase.

Thirty-six delta5- and 5'alpha-3beta-hydroxysteroids have been oxidized with cholesterol oxidase to give the corresponding delta4- and 5alpha-3-ketosteroids, respectively. The mass spectral characteristics of the products (or their trimethylsilyl ether derivatives, in the case of 3-keto-hydroxysteroids) varied considerably, depending especially on the nature of the C-17 sidechain. The ion of m/e 124 (or its equivalent) from cleavage of ring B was frequently a major fragment from delta4-3-ketosteroids, but in some instances was of insignificant abundance. Trimethylsilylation of the product of the oxidation of neoergosterol gave neoergosterone enol-trimethylsilyl ether. Fragmentations of the sidechain predominated in the mass spectra of the 5alpha-3-ketosteroids.

Cholesterol↗
PubMed1982

The presence of 17 beta-hydroxysteroid dehydrogenase activity in preimplantation rat and mouse blastocysts.

When Day 5 rat blastocysts and Day 4 and 5 mouse blastocysts were cultured in 53 microliters of medium containing 1340 or 2680 pg [3H]estradiol (E2), large amounts of [3H]estrone (E1) were detected in the medium at daily intervals for up to 5 days. This indicates the presence of 17 beta-hydroxysteroid dehydrogenase in the embryos. The activity was higher at a higher concentration of E2 and was also higher in mouse than in rat blastocysts. In the mouse, the activity was higher in Day 5 than Day 4 blastocysts during the first day in culture; then it decreased in Day 5 but increased in Day 4 blastocysts. The importance of E2 in embryonic development and implantation as suggested by others may be related to the activity of 17 beta-hydroxysteroid dehydrogenase.

17-Hydroxysteroid Dehydrogenases↗
PubMed1997

The regio- and stereo-selectivity of C19 and C21 hydroxysteroid glucuronidation by UGT2B7 and UGT2B11.

The capacity of two human hepatic UDP-glucuronosyltransferase (UGT) isoforms, UGT2B7 and UGT2B11, to metabolize more than 50 hydroxylated androgens and pregnanes was investigated. All mono- and dihydroxylated androgens with a hydroxyl function in the 3 alpha, 6 alpha, and 17 beta positions were glucuronidated by UGT2B7, but highest activity was generally observed for steroids containing a 3 alpha-hydroxy substituent. UGT2B7 did not glucuronidate 2 alpha-, 2 beta-, 3 beta-, 6 beta-, 7 alpha-, 11 alpha-, and 11 beta-monohydroxylated androgens, although the presence of hydroxyl groups at certain of these positions did not abolish the ability of UGT2B7 to metabolize diols which also possessed a 3 alpha- or 17 beta-hydroxyl group. 3 alpha-Hydroxypregnanes were also glucuronidated by UGT2B7. Activity was not detected for 6 alpha-, 6 beta-, 11 beta-, 12 alpha-, 16 alpha-, 17 alpha-, 20 alpha-, or 21-monohydroxylated pregnanes. Although 11 alpha-hydroxylated androgens were not glucuronidated by UGT2B7, this enzyme exhibited high activity toward the 11 alpha-hydroxylated derivatives of 5 beta-prenanedione and progesterone. UGT2B11 similarly glucuronidated 3 alpha-hydroxyandrogens and -pregnanes, but rates of metabolism were low compared to UGT2B7. With the exception of androsterone and its 5 beta-isomer, ring A/B stereochemistry appeared not to influence rates of hydroxysteroid glucuronidation by UGT2B7 and UGT2B11. Overall, the data indicate a high degree of stereo- and regioselectivity in the glucuronidation of hydroxyandrogens and -pregnanes by UGT2B7 and UGT2B11 and further suggest that UGT2B7 may contribute to the glucuronidation of 3 alpha-hydroxysteroids in humans.

Androgens↗
PubMed2000

17Beta-hydroxysteroid dehydrogenase from Cochliobolus lunatus: model structure and substrate specificity.

A homology-built structural model of 17beta-hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatus, a member of the short-chain dehydrogenase/reductase family, was worked out using the known three-dimensional structure of trihydroxynaphthalene reductase (EC 1.3.1.50) from Magnaporthe grisea as a template. Due to 61% sequence identity, the model also revealed a similar backbone trace. On the basis of qualitative thin-layer chromatography and comparative kinetic tests of the activity toward various potential steroid substrates, we conclude that androgens are more efficiently converted than estrogens. Their specific oxidoreduction predominantly occurs at the C17 position while no significant conversion at C3 and C20 was determined. Additionally, a thousand times effective inhibition by 5-methyl-(1,2,4)-triazolo[3,4-b]benzothiazole and no activity toward 2,3-dihydro-2,5-dihydroxy-4H-benzopyran-4-one indicate distinct specificies of 17beta-hydroxysteroid dehydrogenase from the fungus C. lunatus and trihydroxynaphthalene reductase. The results of the analysis of progress curve measurements for the forward and backward reactions are consistent with the Theorell-Chance reaction mechanism also predicted from the structural model. In accordance with these results, 4-androstene-3,17-dione was docked into the enzyme active site using molecular modeling and dynamics calculations.

17-Hydroxysteroid Dehydrogenases↗
PubMed1977

Ultracytochemical demonstration and probable localization of 3beta-hydroxysteroid dehydrogenase activity with a ferricyanide technique.

In order to localize 3beta-hydroxysteriod dehydrogenase activity on the ultrastructural level, sections of Newt and Rat adrenocortical tissues, fixed in a mixture of glutaraldehyde (0.25%) and formaldehyde (1%), were incubated in a medium containing namely a 3beta-hydroxysteroid as substrate, NAD, potassium ferricyanide as final electron acceptor and copper sulfate. In some experiments, phenazine methosulfate (PMS), an electron carrier which can substitute for the activity of the endogenous NADH-diaphorase, is added at various concentrations to the incubation medium. A final precipitate of copper ferrocyanide is observed in the immediate vicinity of the tubules of the smooth endoplasmic reticulum, or in contact with their external faces. The reaction product can also be seen in mitochondrial cristae. The reaction does not take place in incubation media lacking substrate or containing cyanoketone, a specific inhibitor of 3beta-hydroxysteroid dehydrogenase. the addition of PMS to the incubation medium increases the intensity of the reaction, but does not modify the localization of the precipitate.

Adrenal Glands↗
PubMed1979

A quantitative cytochemical method for the demonstration of delta5,3beta-hydroxysteroid dehydrogenase activity in unfixed tissue sections of rat ovary.

A method for the quantitative measurement of delta5,3beta-hydroxysteroid dehydrogenase activity in unfixed tissue sections of rat ovary has been described. The method depends on the oxidation of dehydroepiandrosterone (DHEA) and uses nitroblue tetrazolium as the final electron acceptor. Although the dehydrogenase is not a soluble enzyme, polyvinyl alcohol is included in the reaction medium to allow the use of a high substrate concentration whilst employing a low concentration (5%) of dimethyl formamide. The enzyme is equally dependent on NAD+ or NADP+ for its activity and this activity is significantly enhanced by the presence of cyanide. The NADP+ dependence is not abolished by inhibiting nonspecific alkaline phomonoesterase. The activity of delta5,3beta-hydroxysteroid dehydrogenase is completely dependent on a functional sulphydryl group. Furthermore, the enzyme activity is totally inhibited in the presence of a steroid substrate analogue at 10(-4) M.

3-Hydroxysteroid Dehydrogenases↗
PubMed1975

[A method for quantitative dertermination of 20-keto steroids in urine during the use of a 3 alpha-, 20 beta-hydroxysteroid: NAD oxidoreductase (author's transl)].

A method is described for the quantitative determination of 20-ketosteroids in urine during the application of a 3alpha, 20beta-hydroxysteroid: NAD oxidoreductase. In the presence of NADH H+ the enzyme catalyses in an "equimolar" developing reaction the reduction of 20-keto groups of C-21 steroids giving 20 beta-alcohols. The measurable NADH H+ consumption is a measure for the quantity of those 20-ketosteroids present in the reaction mixture. The 3alpha-hydroxysteroid: NAD oxidoreductase activity of the enzyme does not influence the specificity of the method to determine the 20-ketosteroids because,-under the present extraction and purification conditions-those 3alpha-hydroxy-5alpha-androstan derivatives are not reaching the reaction mixture of determination. The demands towards the criterions of reliability (correctness and exactitude) come to be fulfilled by this method.

Adolescent↗
PubMed1980

Neutral 17beta-hydroxysteroid oxidoreductase deficiency in testes causing male pseudohermaphroditism in an infant.

A deficiency of neutral 17beta-hydroxysteroid oxidoreductase activity in testes has been diagnosed in a n infant with male pseudohermaphroditism. In vivo stimulation tests of testicular endocrine function with human chorionic gonadotrophin provided an accurate diagnosis in contrast to estimates of enzymic activity in vitro in testes and other tissues. The discrepancy in testes may be due to the absence of gonadotrophin stimulation in the latter studies. The in vitro studies show that there are at least two forms of 17beta-hydroxysteroid oxidoreductase under independent genetic control and that only one form is localized to the testes. The diagnosis before puberty has allowed early treatment by removal of the abnormal testes which should prevent the usual presenting clinical signs of marked masculinizatin and hirsutism at puberty.

17-Hydroxysteroid Dehydrogenases↗
PubMed1992

Mild adrenal 3 beta-hydroxysteroid dehydrogenase deficiency in children with accelerated growth, premature pubarche and/or hirsutism.

We studied eight children (seven girls and one boy, of whom three are siblings) with accelerated growth, premature pubarche and/or hirsutism between the ages of 7 and 9.5 years. These patients were studied for the presence of adrenal steroidogenic defect by adrenocortico tropic hormone (ACTH) stimulation testing (Cortrosyn, 0.25 mg iv bolus dose). Baseline and stimulated 17-hydroxypregnenolone (delta 5-17P), 17-hydroxyprogesterone (17-OHP), dehydroepiandrosterone (DHEA), androstenedione (delta 4-A), testosterone, and cortisol levels were measured. Six children, including three siblings, were diagnosed with a mild form of 3 beta-hydroxysteroid dehydrogenase deficiency based upon the elevated response of serum delta 5-17P and the elevated ratio of delta 5-17P/17-OHP and of DHEA/delta 4-A to ACTH stimulation. Thus, a mild form of 3 beta-hydroxysteroid dehydrogenase deficiency may not be uncommon in children with accelerated growth, premature pubarche and/or hirsutism and routine assay of delta 5-steroid metabolites should be generally available.

17-alpha-Hydroxypregnenolone↗