scholarly journals Cloning and production of antisera to human placental 11 β-hydroxysteroid dehydrogenase type 2

1996 ◽  
Vol 313 (3) ◽  
pp. 1007-1017 ◽  
Author(s):  
Roger W. BROWN ◽  
Karen E. CHAPMAN ◽  
Yuri KOTELEVTSEV ◽  
Joyce L. W. YAU ◽  
Robbie S. LINDSAY ◽  
...  
Keyword(s):  
Reductase Activity ◽  
Hydroxysteroid Dehydrogenase ◽  
Fetal Exposure ◽  
Adult Kidney ◽  
Peptide Carrier ◽  
Affinity Labelling ◽  
Ic50 Values ◽  
20 Nm

By inactivating potent glucocorticoid hormones (cortisol and corticosterone), 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) plays an important role in the placenta by controlling fetal exposure to maternal glucocorticoids, and in aldosterone target tissues by controlling ligand access to co-localized glucocorticoid and mineralocorticoid receptors. Amino acid sequence from homogeneous human placental 11β-HSD2 was used to isolate a 1897 bp cDNA encoding this enzyme (predicted Mr 44126; predicted pI 9.9). Transfection into mammalian (CHO) cells produces 11β-HSD2 activity which is NAD+-dependent, is without reductase activity, avidly metabolizes glucocorticoids (Km values for corticosterone, cortisol and dexamethasone of 12.4±1.5, 43.9±8.5 and 119±15 nM respectively) and is inhibited by glycyrrhetinic acid and carbenoxolone (IC50 values 10–20 nM). Rabbit antisera recognizing 11β-HSD2 have been raised to an 11β-HSD2-(370–383)-peptide–carrier conjugate. Recombinant 11β-HSD2, like native human placental 11β-HSD2, is detectable with affinity labelling and anti-11β-HSD2 antisera, and appears to require little post-translational processing for activity. 11β-HSD2 mRNA (~1.9 kb transcript) is expressed in placenta, aldosterone target tissues (kidney, parotid, colon and skin) and pancreas. In situ hybridization and immunohistochemistry localize abundant 11β-HSD2 expression to the distal nephron in human adult kidney and to the trophoblast in the placenta. 11β-HSD2 transcripts are expressed in fetal kidney (but not lung, liver or brain) at 21–26 weeks, suggesting that an 11β-HSD2 distribution resembling that in the adult is established by this stage in human development.

Intracellular regulation of 17β-hydroxysteroid dehydrogenase type 2 catalytic activity in A431 cells

1997 ◽  
Vol 153 (3) ◽  
pp. 453-464 ◽  
Author(s):  
C H Blomquist ◽  
B S Leung ◽  
C Beaudoin ◽  
D Poirier ◽  
Y Tremblay
Keyword(s):  
Reductase Activity ◽  
Maximum Velocity ◽  
Hydroxysteroid Dehydrogenase ◽  
A431 Cells ◽  
Intact Cells ◽  
Cell Monolayers ◽  
Intracellular Regulation

Abstract There is growing evidence that various isoforms of 17β-hydroxysteroid dehydrogenase (17-HSD) are regulated at the level of catalysis in intact cells. A number of investigators have proposed that the NAD(P)/NAD(P)H ratio may control the direction of reaction. In a previous study, we obtained evidence that A431 cells, derived from an epidermoid carcinoma of the vulva, are enriched in 17-HSD type 2, a membrane-bound isoform reactive with C18 and C19 17β-hydroxysteroids and 17-ketosteroids. The present investigation was undertaken to confirm the presence of 17-HSD type 2 in A431 cells and to assess intracellular regulation of 17-HSD at the level of catalysis by comparing the activity of homogenates and microsomes with that of cell monolayers. Northern blot analysis confirmed the presence of 17-HSD type 2 mRNA. Exposure of cells to epidermal growth factor resulted in an increase in type 2 mRNA and, for microsomes, increases in maximum velocity (Vmax) with no change in Michaelis constant (Km) for testosterone and androstenedione, resulting in equivalent increases in the Vmax/Km ratio consistent with the presence of a single enzyme. Initial velocity data and inhibition patterns were consistent with a highly ordered reaction sequence in vitro in which testosterone and androstenedione bind only to either an enzyme–NAD or an enzyme–NADH complex respectively. Microsomal dehydrogenase activity with testosterone was 2- to 3-fold higher than reductase activity with androstenedione. In contrast, although cell monolayers rapidly converted testosterone to androstenedione, reductase activity with androstenedione or dehydroepiandrosterone (DHEA) was barely detectable. Lactate but not glucose, pyruvate or isocitrate stimulated the conversion of androstenedione to testosterone by monolayers, suggesting that cytoplasmic NADH may be the cofactor for 17-HSD type 2 reductase activity with androstenedione. However, exposure to lactate did not result in a significant change in the NAD/NADH ratio of cell monolayers. It appears that within A431 cells 17-HSD type 2 is regulated at the level of catalysis to function almost exclusively as a dehydrogenase. These findings give further support to the concept that 17-HSD type 2 functions in vivo principally as a dehydrogenase and that its role as a reductase in testosterone formation by either the Δ4 or Δ5 pathway is limited. Journal of Endocrinology (1997) 153, 453–464

Nutritional marginal zinc deficiency disrupts placental 11β-hydroxysteroid dehydrogenase type 2 modulation

2016 ◽  
Vol 7 (1) ◽  
pp. 84-92 ◽  
Author(s):  
Y. L. Huang ◽  
S. Supasai ◽  
H. Kucera ◽  
N. W. Gaikwad ◽  
A. M. Adamo ◽  
...  
Keyword(s):  
Zinc Deficiency ◽  
Hydroxysteroid Dehydrogenase ◽  
Fetal Exposure ◽  
Marginal Zinc Deficiency ◽  
Zinc Nutrition

This paper investigated if marginal zinc nutrition during gestation could affect fetal exposure to glucocorticoids as a consequence of a deregulation of placental 11βHSD2 expression.

Proinflammatory cytokines inhibit human placental 11β-hydroxysteroid dehydrogenase type 2 activity through Ca2+ and cAMP pathways

2006 ◽  
Vol 290 (2) ◽  
pp. E282-E288 ◽  
Author(s):  
Iren Kossintseva ◽  
Susan Wong ◽  
Ed Johnstone ◽  
Larry Guilbert ◽  
David M. Olson ◽  
...  
Keyword(s):  
Cardiovascular Disease ◽  
Adenylyl Cyclase ◽  
Proinflammatory Cytokines ◽  
Hydroxysteroid Dehydrogenase ◽  
Mrna Levels ◽  
Fetal Exposure ◽  
Increased Risk ◽  
Tnf Α ◽  
Intracellular Ca

Excessive fetal exposure to glucocorticoids has been implicated in the etiology of adult metabolic and cardiovascular disease. Placental 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) may protect the fetus from excessive glucocorticoid exposure. Maternal stress may be accompanied by elevated levels of cortisol and increased proinflammatory cytokines [interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNF-α)]. We hypothesize that proinflammatory cytokines inhibit human placental 11β-HSD activity. We incubated explant cultures of term human placental villi in the presence or absence of 10 ng/ml IL-1β, IL-6, or TNF-α, with or without agonists or antagonists of intracellular Ca2+ and adenylyl cyclase. Activity for 11β-HSD2 was estimated using a radioisotope assay, and mRNA was measured using quantitative RT-PCR. All cytokines significantly ( P ≤ 0.05) reduced 11β-HSD2 activity (>75% suppression); maximal inhibition occurred within 2 h and was maintained for at least 24 h. The IL-1β-induced inhibitory activity was attenuated using a Ca2+ channel blocker (nifedipine), an intracellular Ca2+ antagonist [8-( N, N-diethylamino)octyl-3,4,5-trimethoxybenzoate], or the adenylyl cyclase stimulant forskolin. Conversely, 11β-HSD2 activity was diminished in the presence of the Ca2+ ionophore A-23187 or the adenylyl cyclase inhibitor SQ-22536. mRNA levels for 11β-HSD2 were not changed by any of the treatments. Proinflammatory cytokines inhibit human placental 11β-HSD2 activity through a mechanism that involves increased intracellular Ca2+ and inhibition of adenylyl cyclase. This could result in excessive fetal exposure to maternal cortisol. This mechanism might mediate part of the increased risk of metabolic and cardiovascular disease in adult offspring.

Developmental and glucocorticoid regulation of pituitary 11 β-hydroxysteroid dehydrogenase 1 gene expression in the ovine fetus and lamb

1995 ◽  
Vol 14 (1) ◽  
pp. 109-116 ◽  
Author(s):  
K Yang ◽  
S G Matthews ◽  
J R G Challis
Keyword(s):  
Reductase Activity ◽  
Hydroxysteroid Dehydrogenase ◽  
Postnatal Period ◽  
Pars Intermedia ◽  
Pars Distalis ◽  
Short Term ◽  
Fetal Sheep ◽  
Ovine Fetus

ABSTRACT To examine the role of 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) in the control of glucocorticoid actions in the ovine pituitary during development, we have sought developmental changes in the distribution and the level of 11β-HSD1 mRNA by in situ hybridization. In the pars distalis, 11β-HSD1 mRNA was present by day 60; its amount did not change significantly until term (days 145–147) when it increased dramatically. The level of 11β-HSD1 mRNA increased further during the postnatal period. In contrast, 11β-HSD1 mRNA in the pars intermedia was not detectable until day 135; it increased in amount at days 140–143, but did not change significantly thereafter through to adulthood. We have also measured levels of both dehydrogenase and reductase activities of 11β-HSD1 in the pars distalis of fetal sheep at day 140 and term, and of postnatal sheep at 1–2 months of age, to determine whether changes in 11β-HSD1 mRNA are reflected in the levels of enzyme activities. There were progressive increases in both dehydrogenase and reductase activities from day 140 to 1–2 months postnatally, although dehydrogenase activity was consistently higher than reductase activity. Finally, we have determined the effect of short-term intrafetal cortisol infusion (5 μg/min for 12 h) on levels of pituitary 11β-HSD1 mRNA by in situ hybridization. There was no effect of cortisol infusion on 11β-HSD1 mRNA expression. The present results demonstrate that 11β-HSD mRNA and enzyme activity in the pars distalis of fetal sheep increase dramatically at term when plasma levels of both ACTH and cortisol are elevated. This suggests that 11β-HSD1 may contribute to the proposed resetting of cortisol negative feedback within the fetal pituitary at that time.

scholarly journals Purification of 11 β-hydroxysteroid dehydrogenase type 2 from human placenta utilizing a novel affinity labelling technique

1996 ◽  
Vol 313 (3) ◽  
pp. 997-1005 ◽  
Author(s):  
Roger W. BROWN ◽  
Karen E. CHAPMAN ◽  
Parvez MURAD ◽  
Christopher R. W. EDWARDS ◽  
Jonathan R. SECKL
Keyword(s):  
Human Placenta ◽  
Pressure Control ◽  
Hydroxysteroid Dehydrogenase ◽  
Distal Nephron ◽  
Tissue Extracts ◽  
Affinity Labelling ◽  
Short Chain Alcohol ◽  
Internal Amino Acid Sequence ◽  
Dimensional Electrophoresis

11β-Hydroxysteroid dehydrogenase type 2 (11β-HSD2) efficiently inactivates potent glucocorticoid hormones (cortisol and corticosterone), leaving aldosterone unmetabolized. Abundant 11β-HSD2 activity in human placenta plays a central role in controlling fetal glucocorticoid exposure, which if excessive is harmful and may predispose to low birth weight and hypertension in adulthood. Similar 11β-HSD2 activity in the distal nephron protects mineralocorticoid receptors from glucocorticoids and appears to be important in normal blood pressure control. We have purified human placental 11β-HSD2 16000-fold, to homogeneity, and determined over 100 residues of the internal amino acid sequence. Purification was assisted by a novel technique allowing highly specific (single spot on two-dimensional electrophoresis) photoaffinity labelling of active 11β-HSD2 in crude tissue extracts by its glucocorticoid substrates. This work reveals that 11β-HSD2 is a member of the short-chain alcohol dehydrogenase superfamily (apparent monomer Mr ~40000). It is a very basic (apparent pI = 9.1) intrinsic membrane protein, requiring as yet undefined membrane constituents for full stability. Affinity chromatography and affinity labelling studies suggest that 11β-HSD2 has a compulsory ordered mechanism, with NAD+ binding first, followed by a conformational change allowing glucocorticoid binding with high affinity.

In-Situ Observation of Clay Minerals Hydrated and Intercalated With Liquid Chemicals Using Film-Sealed Environmental Cell

1980 ◽  
Vol 38 ◽  
pp. 208-209 ◽  
Author(s):  
K. Fukushima ◽  
N. Kohyama ◽  
A. Fukami
Keyword(s):  
Carbon Film ◽  
Resolving Power ◽  
In Situ Observation ◽  
Interlayer Water ◽  
Saturated Water ◽  
Structure Changes ◽  
Environmental Cell ◽  
Gas Layer ◽  
20 Nm

A film-sealed high resolution environmental cell(E.C) for observing hydrated materials had been developed by us(l). Main specification of the E.C. is as follows: 1) Accelerated voltage; 100 kV. 2) Gas in the E.C.; saturated water vapour with carrier gas of 50 Torr. 3) Thickness of gas layer; 50 μm. 4) Sealing film; evaporated carbon film(20 nm thick) with plastic microgrid. 5) Resolving power; 1 nm. 6) Transmittance of electron beam; 60% at 100 kV. The E.C. had been successfully applied to the study of hydrated halloysite(2) (3). Kaolin minerals have no interlayer water and are basically non-expandable but form intercalation compounds with some specific chemicals such as hydrazine, formamide and etc. Because of these compounds being mostly changed in vacuum, we tried to reveal the structure changes between in wet air and in vacuum of kaolin minerals intercalated with hydrazine and of hydrated state of montmori1lonite using the E.C. developed by us.

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