Biomedical subjects

A Marandici

Publications and source records attributed to A Marandici.

15 recordsLinked to original sources

PubMed1997

Hormonal regulation of oxidative and reductive activities of 11 beta-hydroxysteroid dehydrogenase in rat Leydig cells.

We have proposed that the 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) of Leydig cells protects against glucocorticoid-induced inhibition of testosterone (T) production. However, Leydig cells express type I 11 beta-HSD, which has been shown to be reductive in liver parenchymal cells. Because reduction would have the opposite effect of activating glucocorticoid, the present study was designed to determine: 1) whether Leydig cell 11 beta-HSD is primarily oxidative or reductive; and 2) whether oxidative and reductive activities are separately modified by known regulators of Leydig cell steroidogenic function. Leydig cells and liver parenchymal cells were purified from mature male Sprague-Dawley rats (250 g BW), and 11 beta-HSD oxidative and reductive activities were measured using radiolabeled substrates and TLC of triplicate media samples from 1-h incubations immediately after cell isolation. Enzyme activities also were examined in purified Leydig cells at the end of 3 days of culture in vitro in the presence of LH (10 ng/ml), dexamethasone (DEX, 100 nM), T (50 nM), or epidermal growth factor (EGF, 50 ng/ml). In confirmation of previous reports, the reductive activity of 11 beta-HSD was predominant over oxidation in liver parenchymal cells. In contrast, 11 beta-HSD oxidative activity prevailed over reduction in Leydig cells by a ratio of 2:1. The activities of 11 beta-HSD also were analyzed in Leydig cells that were purified 7 days after endogenous glucocorticoid levels were suppressed by adrenalectomy (ADX). Oxidative activity declined in Leydig cells after ADX (22.53 +/- 1.12 pmol/h.10(6) cells, mean +/- SEM vs. 31.47 +/- 1.48 pmol/.10(6) cells in sham-operated controls, P < 0.05), whereas there was no change in reductive activity. This indicated that physiologically active corticosterone is involved in maintaining the predominance of 11 beta-HSD oxidation. When enzyme activities were analyzed in Leydig cells after 3 days of hormonal treatment in vitro, oxidation and reduction were observed to change in opposing directions. Culture of Leydig cells from sham-operated control rats with either LH, T, or EGF resulted in declines in oxidative activity from 33.35 +/- 0.77 to 28.24 +/- 1.93, 27.30 +/- 0.96, and 24.13 +/- 1.02 pmol/ h.10(6) cells (x +/- SE), respectively. However, EGF stimulated 11 beta-HSD reductive activity in cultured Leydig cells from both control (from 18.97 +/- 1.10 to 27.16 +/- 0.71 pmol/h.10(6) cells and ADX rats (from 16.51 +/- 0.75 to 23.56 +/- 0.84 pmol/h.10(6) cells). Among the hormonal treatments, only DEX increased oxidative activity and simultaneously decreased reductive activity in Leydig cells from ADX rats. This increase accentuated the predominance of oxidative activity in Leydig cells, with a ratio of oxidative to reductive activity of 4:1 after DEX treatment, compared with 2:1 in controls that were untreated. We conclude that 11 beta-HSD activity in Leydig cells is primarily oxidative. Moreover, oxidation and reduction are regulated separately by hormones.

11-beta-Hydroxysteroid Dehydrogenases↗

PubMed1994

11 beta-Hydroxysteroid dehydrogenase in the rat inner ear.

11 beta-Hydroxysteroid dehydrogenase (11 beta-HSD) was demonstrated specifically in the spiral ligament of the cochlear membranous labyrinth by enzyme assay, Western blotting, and immunocytochemistry. Other cochlear regions and the vestibular membranous labyrinth were devoid of 11 beta-HSD. Spiral ligament 11 beta-HSD exerted predominantly an oxidative activity and was NADP specific, which is similar to 11 beta-HSD in most other tissues. 11 beta-HSD was colocalized with mineralocorticoid and glucocorticoid steroid receptors in the spiral ligament. 11 beta-HSD may control steroid binding to these inner ear steroid receptors and, in addition, may regulate steroid receptor binding in the adjacent stria vascularis in paracrine fashion.

11-beta-Hydroxysteroid Dehydrogenases↗

PubMed1994

11 beta-Hydroxysteroid dehydrogenase alleviates glucocorticoid-mediated inhibition of steroidogenesis in rat Leydig cells.

Leydig cells from mature rat testes contain high levels of 11 beta-hydroxysteroid dehydrogenase (11HSD), an enzyme that oxidatively inactivates glucocorticoids. We have proposed that the 11HSD of Leydig cells protects the testis from the effects of high levels of glucocorticoids, as may occur in stress and Cushing's disease. In this paper we investigate whether testicular 11HSD by inactivating glucocorticoids diminishes their ability to inhibit testosterone (T) production. Corticosterone (B) and dexamethasone (DEX) inhibited T production by purified Leydig cells in a dose-dependent manner. Activity was diminished by 50% with 1.5 nM DEX vs. 0.4 microM B. The shapes of the inhibition curves were consistent with a saturable process; inhibition by both steroids was overcome with the glucocorticoid receptor antagonist RU486. We concluded that the effect was mediated by glucocorticoid receptors. Aldosterone, 11 beta-hydroxyprogesterone, and 11-deoxycorticosterone did not decrease T production. The greater potency of DEX compared to B may be due to its resistance to oxidative inactivation by 11HSD. As 11-dehydrocorticosterone, the product of the oxidation of B by 11HSD, did not inhibit T production, it was predicted that inactivation of 11HSD should enhance the inhibitory effect of B. Consistent with this prediction, inhibition by B was increased by carbenoxolone, an inhibitor of 11HSD, becoming more similar to that by DEX. Suppression of T production by DEX (which is not a substrate of 11HSD) was unaffected by carbenoxolone. We conclude that through reduction of the levels of inhibitory glucocorticoids, 11HSD has a novel role among Leydig cell steroid-metabolizing enzymes in the regulation of T production.

11-beta-Hydroxysteroid Dehydrogenases↗

PubMed1993

Inhibition by glycyrrhetinic acid of rat tissue 11 beta-hydroxysteroid dehydrogenase in vivo.

Inhibition of 11 beta-hydroxysteroid dehydrogenase (11-HSD) in the major organs of the rat by the inhibitor glycyrrhetinic acid (a component of licorice) was investigated. The inhibitor decreased 11-HSD levels in most organs after intraperitoneal injection. The effect was dose dependent, was maximally effective 3 hours after administration, and was completely reversed at 24 hours. The magnitude and patterns of effectiveness differed between tissues. It was concluded that the inhibition of 11-HSD by glycyrrhetinic acid is not restricted to one or a few organs, but is broadly based.

11-beta-Hydroxysteroid Dehydrogenases↗

PubMed1991

Corticosteroid side-chain isomerase in the circulatory system.

Corticosteroid side-chain (CSC) isomerase catalyzes ketol-aldol interconversion of the corticosteroid side chain. The enzyme was present in the blood of mouse, rat, guinea pig, chicken, pig, horse, sheep, cow, and human. The patterns of substrate specificity, measuring 3H-1H exchange of 21-tritiated forms of 11-deoxycorticosterone, corticosterone, and cortisol, were species specific. Based on enzyme activity and immunostaining of mouse blood fractions, red blood cells had the most isomerase activity, plasma had less, and white blood cells had low but highly variable levels of enzyme. Purified mouse liver CSC isomerase was found to be adsorbed by red blood cells. The results suggest that circulating CSC isomerase is derived in part from tissue sources and is in part an intrinsic blood enzyme.

Animals↗

PubMed1990

Purification and characterization of corticosteroid side chain isomerase.

Corticosteroid side chain isomerase of rat liver catalyzes the interconversion of the ketol (20-oxo-21-ol) and aldol (20-hydroxy-21-al) forms of the corticosteroid side chain. The enzyme has now been purified to apparent homogeneity from rat liver cytosol by sequential chromatography on anionic, hydroxylapatite, and gel filtration columns. Ketol-aldol isomerization is followed by measuring the exchange of tritium from 21-tritiated steroids with water. The native enzyme is a dimer of MW 44,000. The isoelectric point is 4.8 +/- 0.1 pH units. The purified enzyme is stimulated by Co3+ or Ni2+. The enzyme utilizes 11-deoxycorticosterone, corticosterone, and 17-deoxycortisol as substrate but not cortisol, tetrahydrocortisol, and prednisolone. Tritium-water exchange of (21S)-[21-3H]DOC is a pseudo-first-order reaction; 21-3H exchange from the 21R isomer proceeds with first-order kinetics only after a lag associated with its epimerization to the 21S form.

Amino Acids↗

PubMed1990

Corticosteroid side-chain isomerase in mouse organs: kinetic and immunologic studies.

We have investigated the distribution of corticosteroid side-chain (CSC) isomerase in the tissues of mice using as criteria its enzyme activity and immunoreactivity with monospecific polyclonal antibodies generated in rabbits. CSC isomerase was present in all organs examined. The liver and kidney contained the highest activity. The strain-dependent differences that we had previously reported for liver (i.e., BALB/c greater than C57BL/6) extended to the other organs, including the kidney, brain, heart, muscle, pancreas, testis, thymus, and lung. Western blot analysis showed a single antigen, identical in all tissues, corresponding in mobility to purified CSC isomerase. The intensities of the bands were generally proportional to enzyme activities. Titration of homogeneous enzyme with the IgG fraction of antiserum (unfractionated serum had some CSC isomerase activity) caused an increase in activity, followed by rapid inactivation after the addition of more antiserum. The broad distribution of CSC isomerase suggests that the ketol-aldol interconversion of the CSC may play a role other than, or in addition to, initiating metabolic inactivation of corticosteroids.

Animals↗

PubMed1989

Synthesis of tritium labeled cortoic acids.

A procedure is described for the microsynthesis and purification of the high specific activity tritium labeled cortisol metabolites, 20 alpha- and 20 beta-cortolic acids and 20 alpha- and 20 beta-cortolonic acids.

Cortisone↗

PubMed1986

Estrogen and progesterone receptors in the organs of prenatal cynomolgus monkey and laboratory mouse.

The estrogen and progesterone receptors of several organs of the prenatal cynomolgus macaque and the fetal mouse were studied using a combination of the dextran-coated charcoal technique and high-performance liquid chromatography. This procedure permitted the concurrent measurement of both receptors in minute amounts of tissue. Estrogen receptors, but not progesterone receptors, were found in the fetal monkey and mouse uteri. No estrogen or progesterone receptors were detected in the lungs, liver, kidney, heart, brain, adrenal gland, or limbs of mouse or monkey fetuses. The nonspecific binding of radioactive ORG-2058 was not displaced by unlabeled progesterone, 17 alpha-hydroxyprogesterone caproate, or ORG-2058. Because the steroid receptors that are indispensable mediators of steroid hormone action were absent from the nonreproductive tissues, prenatal development of these organs and tissues cannot be adversely influenced by exposure to estradiol, progesterone, or their synthetic analogues.

17 alpha-Hydroxyprogesterone Caproate↗

PubMed1985

Comparison of the distribution kinetics and metabolism to acid end-products of corticosterone and 11-deoxycorticosterone in BALB/c mice.

The conversion of [4 14C]corticosterone[( 14C]B) and 11-deoxy-[1,2-3H]corticosterone [( 3H]DOC) to steroidal carboxylic acids was studied in the BALB/c mouse. There was rapid and preferential excretion of [3H]DOC metabolites into the gastrointestinal tract. Excretion of 14C through the kidney was higher than 3H excretion. Within minutes of intraperitoneal injection, levels of 3H and 14C in most organs reached their maximal levels and subsequently decreased in an exponential pattern. The majority of the organs took up 14C to a greater extent than 3H. Using tissue blood ratio of tracer (T/B) as criterion, it was found that liver, gall bladder, intestine, and kidney concentrated 3H and 14C-labeled steroid from blood. T/B for 3H exceeded that for 14C in the gastrointestinal tract. Abdominal fat preferentially took up [3H]DOC tracer, whereas [14C]B tracer was not taken up by this tissue. T/B was less than 1 for 3H and 14C in heart, thymus, spleen, brain, skeletal muscle and skin. In these organs uptake of B and its metabolites was greater than that of DOC and its metabolites. In liver, [14C]B and [3H]DOC were converted to carboxylic acid metabolites which accumulated in the intestine. The most abundant acid was 11 beta,20 alpha-dihydroxy-3-oxo-pregn-4-en-21-oic acid from B. The acid metabolites of DOC were not identified. For both steroids, acids were major metabolic end-products.

Animals↗

PubMed1984

The fate of corticosterone and 11-deoxycorticosterone in C57BL/6 and BALB/c strains of mice: distribution and oxidative metabolism.

The distribution kinetics and oxidative metabolism of [4-C14] corticosterone (B) and 11-deoxy-[1,2-3H] corticosterone (DOC) were compared in C57BL/6 (B6) and BALB/c (C) mice. Statistically important differences in the distribution of [14C]B and [3H]DOC occurred that were independent of strain, while other differences were strain dependent. Intestinal excretion of metabolites of B and DOC was greater in B6 mice than in C mice, and kidney excretion was greater in C mice than B6 mice. In both C and B6 mice, 3H was cleared from liver faster than 14C, with no strain differences. DOC metabolite levels exceeded B metabolite levels in small intestine and gall bladder of both strains. In most other organs, B metabolites exceeded DOC metabolites. Time average strain differences in accumulation of B and its metabolites favoring B6 were found in pancreas, brain, lung, heart, muscles, adrenals, spleen, mesentery and small intestine. Except for the organs of excretion, no strain differences were found for [3H]DOC metabolites. Sixty minutes after steroid administration, 45% of B metabolites and a third of DOC metabolites were 20-hydroxy-21-oic acids. In the intestine, accumulation of acids derived from either B or DOC was greater for B6 than C strain mice, reflecting the greater proportion of total steroid excreted in the B6 strain.

Animals↗

PubMed1983

Metabolism of corticosterone in the mouse. Identification of 11 beta, 20 alpha-dihydroxy-3-oxo-4-pregnen-21-oic acid as a major metabolite.

We have shown that mouse liver contains enzymes that catalyze the conversion of the ketol side chain to the 20-hydroxy-21-oic acid side chain. In this paper, we have studied the oxidative metabolism of corticosterone to acidic end products in intact mice. A significant fraction of radioactivity from intraperitoneal injections of [4-14C]corticosterone appeared in liver and intestine within 5 min. The major steroid in liver at 5 min postinjection was found to be corticosterone, although acidic metabolites were detected. Within 30 min after injection, 11 beta, 20 alpha-dihydroxy-3-oxo-preg-4-en-21-oic acid became the dominant steroid. At 60 min, it was the major steroid isolated from liver or intestine. Several other acid metabolites were present in lesser amounts in both organs. About half of the remaining radioactive metabolites in liver and intestine were steroid acids, as determined by their reaction with diazomethane. The identification of the major steroid acid as 11 beta, 20 alpha-dihydroxy-3-oxo-pregn-4-en-21-oic acid was made by comparing the chromatographic behavior of the free acid and its methyl ester with that of authentic synthetic acid using thin layer and high performance liquid chromatography. Identity was confirmed by showing that the specific activities of the homogeneous 14C-labeled free acid remained unchanged when reanalyzed as the 21-methyl ester.

Animals↗

PubMed1983

Genetic control of corticosteroid side-chain isomerase activity in the mouse.

The corticosteroid side-chain isomerases of mammalian liver catalyze the interconversion of the ketol and aldol side chains. In the mouse, isomerase was low in C57BL/6 (B6) mice (130 pmol/mg protein . 2 h) and high in BALB/c (C) mice (230 pmol/mg protein . 2 h). From analysis of hybrids between B6 and C and of backcrosses of these hybrids to B6, it was concluded that isomerase levels are controlled by a single autosomal gene dominant for high activity. The distribution of high and low isomerase levels in a series of CXB/By recombinant inbred strains of mice was consistent with linkage of the isomerase gene to H-2. Congenic BALB.B mice (H-2b haplotype from C57BL/10) had low isomerase activities corresponding to C57BL/10, not the high activity of the background strain BALB/c(H-2d). Similarly, BN10.D2 congenic mice (H-2d haplotype from the DBA/2 strain) had high activity characteristic of DBA/2. In the (C X B6)F1, (C X BALB.B)F1 and (B10 X B10.D2)F1 hybrids, all of which are H-2d/H-2d heterozygotes, isomerase activity was high. The association of isomerase levels with H-2 type was further confirmed in mice of the following backcrosses: (C X BALB.B)F1 X BALB.B, (C X B6)F1 X B6 and (B10 X B10.D2)F1 X B10. H-2b/H-2b homozygous segregants had consistently low activity and H-2b/H-2d heterozygous segregants had consistently high activity. It was concluded that the level of corticosteroid side-chain isomerase activity in mouse liver is controlled by a gene(s) in the region of the H-2 locus on chromosome 17.

Animals↗

PubMed1983

A comparison of the tissue distribution and metabolism of 11-deoxy-[1,2-3H]corticosterone in the BALB/c and C57BL/6 strains of mice.

Corticosteroid side-chain isomerase of mouse liver catalyzes the reversible interconversion of the ketol and aldol configurations of the corticosteroid side chain. Activity of the enzyme is under genetic control. To see if the differences in activity that were observed in vitro between inbred strains of mice were also expressed in vivo, the metabolism of 11-deoxy-[1,2-3H]corticosterone ([1,2-3H]DOC) was studied in BALB/c (C) and C57BL/6 (B6) mice. Maximum radioactivity appeared in most organs within 5-10 min after ip injection. Uptake of tracer into liver was greater for C than B6 mice. Tritium levels in blood, kidney, and pancreas were higher in C mice; levels in adrenal, abdominal fat, and mesentery were higher in B6 mice. In both strains, the concentrations of tracer in tissues, except in gastrointestinal tract, declined and reached a minimum within 60 min. Most of the radioactivity (84%) from [1,2-3H]DOC accumulated in the lumen of the intestinal tract, and few counts were found in the wall. Intestinal concentrations of 3H at different postinjection intervals were greater for B6 than C mice. In contrast, twice as much radioactivity appeared in the kidneys of C than of B6 mice. The organs of excretion (kidney, liver, gall bladder, and intestine) concentrated steroid from blood. Heart, striated muscle, and spleen excluded steroid. Four acidic metabolites of [1,2-3H]DOC were detected in liver, and two were detected in small intestine. Acids formed in liver did not accumulate, and no differences between C and B6 strains were seen. More acid metabolites accumulated in intestines of C mice than in those of B6 mice. The quantitative aspects of steroid acid formation in vivo are consistent with our previous in vitro findings that livers from C mice synthesize more pregnolic acid from DOC than do livers from B6 mice.

Acids↗

PubMedDate not supplied

Corticosteroid-calcium complexes.

Glucocorticoids and calcium ions are shown to interact to yield a complex with properties that are distinct from those of the reactants. Reaction of steroids with Ca2+ appears to require the dihydroxyacetone side chain, since other structures do not react. Evidence for complex formation are: increased aqueous solubility of cortisol when Ca2+ is added to an aqueous or a biphasic aqueous/chloroform (or ethyl acetate) system; increased rate of migration of cortisol during reversed-phase thin-layer chromatography and HPLC; chromatographic comigration of 45Ca2+ and 3H-labeled cortisol; coprecipitation of 45Ca2+-3H-cortisol complexes. After dissociation of the cortisol-calcium complex, the only steroid recovered was cortisol. By the above criteria, the properties of cortisol were not affected by Sr2+, Ba2+, or Mg2+. The cleavage patterns of cortisol in the mass spectrometer corresponded to that of 11 beta-hydroxyandrostenedione when Ca2+ was present, and to cortisol in its absence. We therefore postulate that the structure of the dihydroxyacetone side chain was transiently altered by Ca2+, resulting in a labile C17-C20 bond. These results support our earlier proposal that the chemical and physico-chemical properties of corticosteroids are modified by calcium ions.

Adrenal Cortex Hormones↗