Metabolism and receptor binding of nandrolone and testosterone under in vitro and in vivo conditions

1985 ◽  
Vol 110 (3_Suppla) ◽  
pp. S31-S37 ◽  
Author(s):  
E. W. Bergink ◽  
J. A. A. Geelen ◽  
E. W. Turpijn
Keyword(s):  
Androgen Receptor ◽  
Receptor Binding ◽  
Reductase Activity ◽  
Hydroxysteroid Dehydrogenase ◽  
In Vivo Studies ◽  
Muscular Tissue ◽  
Binding Studies ◽  
Tissue Homogenates

Abstract. The metabolism and receptor binding of nandrolone (N) and testosterone (T) were studied under in vitro and in vivo conditions. The results of both in vitro incubation studes with 3H-N and 3H-T in tissue homogenates from rats and in vivo infusion studies with 3H-N and 3H-T in conscious rats show the importance of the enzymes 5α-reductase and 3α/β-hydroxysteroid-oxidoreductases in the prostate and the importance of the enzyme 17β-hydroxysteroid dehydrogenase in the kidney for the effects of N and T on these tissues. Following infusion of a combined dose of 3H-N and 3H-T there is a preferential retention at the receptor of 5α-dihydrotestosterone (DHT) over 5α-dihydronandrolone (DHN), N and T (DHT ⪢ DHN > N > T) in the prostate because T is a better substrate than N for 5α-reductase and because DHT binds more strongly to the androgen receptor than DHN, N and T. In the kidney 5α-reductase is not important; there is a preferential retention of N in T (DHN and DHT were only present in small amounts) because N is less susceptible than T for metabolic inactivation by the enzyme 17β-hydroxysteroid dehydrogenase and N binds strongly to the androgen receptor. Both in vitro and in vivo studies show that N and T were relatively stable in spleen, thymus and muscular tissue (only shown in vivo) and, as a result, the same amount of N and T was bound to the receptor in these tissues in the in vivo infusion experiment. In vitro binding studies with the androgen receptor in intact human cells show that 5α-reduction increases the affinity of T and decreases the affinity of N and of the 17α-ethyl derivative of N (3-keto-ethylestrenol). The results of the present studies explain the relatively strong effect of N, or derivatives of N, compared to that of T on tissues devoid of 5α-reductase activity (e.g. muscular tissue) and they suggest that in particular there may be a strong effect of N on tissues which in addition have a high 17β-hydroxysteroid dehydrogenase activity (e.g. kidney).

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

Hormonal regulation of canine intestinal cholesterol synthesis

1981 ◽  
Vol 240 (4) ◽  
pp. G274-G280
Author(s):  
M. W. Goodman ◽  
W. F. Prigge ◽  
R. L. Gebhard
Keyword(s):  
Cyclic Nucleotides ◽  
Hormonal Regulation ◽  
Cholesterol Synthesis ◽  
Reductase Activity ◽  
In Vivo Studies ◽  
Synthesis Rate ◽  
Ileal Mucosa ◽  
Control Activity

Hormonal regulation of intestinal cholesterol synthesis was studied both in vitro and in vivo. Cholesterol synthesis rate was determined by measurement of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (EC 1.1.1.34) activity and by incorporation [14C]acetate into sterol. In vitro studies utilized organ culture of canine ileal mucosa. During 6-h culture, reductase activity was stimulated sevenfold. Insulin (10-6 M) augmented this rise to 144 +/- 7% of th control activity, while 10(-8) M glucagon, 10(-3) M adenosine 3',5'-cyclic monophosphate, and 3-isobutyl-1-methylxanthine suppressed activity (final reductase activity was 83 +/- 3%, 75 +/- 4%, and 41 +/- 3%, respectively, of cultured control values). In vivo studies utilized dogs with isolated Thiry-Vella ileal fistulas. In vivo, insulin doubled reductase activity while glucagon led to a 42 +/- 9% suppression. It is concluded that insulin and glucagon may be potential physiological regulators of intestinal cholesterol synthesis. The glucagon effect may be mediated by cyclic nucleotides.

In Vitro Androgen Receptor Binding Affinity and in Vivo Inhibitory Activity of 5?-Pregnane-3, 20-Dione

1988 ◽  
Vol 529 (1 Fourth Colloq) ◽  
pp. 239-241
Author(s):  
SAUDHAMINI PARTHASARATHY ◽  
ANDREA CHIN ◽  
VIRGINIA MALLOY ◽  
JONATHAN MATIAS
Keyword(s):  
Androgen Receptor ◽  
Binding Affinity ◽  
Receptor Binding ◽  
Inhibitory Activity ◽  
Receptor Binding Affinity

scholarly journals Modulation of 11β-Hydroxysteroid Dehydrogenase Isozymes by Growth Hormone and Insulin-Like Growth Factor: In Vivo and In Vitro Studies1

1999 ◽  
Vol 84 (11) ◽  
pp. 4172-4177 ◽  
Author(s):  
J. S. Moore ◽  
J. P. Monson ◽  
G. Kaltsas ◽  
P. Putignano ◽  
P. J. Wood ◽  
...  
Keyword(s):  
Growth Factor ◽  
Reductase Activity ◽  
Primary Cultures ◽  
Inverse Correlation ◽  
Hydroxysteroid Dehydrogenase ◽  
Insulin Like Growth Factor ◽  
Igf I ◽  
Glucose Output

The interconversion of hormonally active cortisol (F) and inactive cortisone (E) is catalyzed by two isozymes of 11β-hydroxysteroid dehydrogenase (11βHSD), an oxo-reductase converting E to F (11βHSD1) and a dehydrogenase (11βHSD2) converting F to E. 11βHSD1 is important in mediating glucocorticoid-regulated glucose homeostasis and regional adipocyte differentiation. Earlier studies conducted with GH-deficient subjects treated with replacement GH suggested that GH may modulate 11βHSD1 activity. In 7 acromegalic subjects withdrawing from medical therapy (Sandostatin-LAR; 20–40 mg/month for at least 12 months), GH rose from 7.1 ± 1.5 to 17.5 ± 4.3 mU/L (mean ± se), and insulin-like growth factor I (IGF-I) rose from 43.0 ± 8.8 to 82.1 ± 13.7 nmol/L (both P < 0.05) 4 months after treatment. There was a significant alteration in the normal set-point of F to E interconversion toward E. The fall in the urinary tetrahydrocortisols/tetrahydocortisone ratio (THF+allo-THF/THE; 0.82 ± 0.06 to 0.60 ± 0.06; P < 0.02) but unaltered urinary free F/urinary free E ratio (a marker for 11βHSD2 activity) suggested that this was due to inhibition of 11βHSD1 activity. An inverse correlation between GH and the THF+allo-THF/THE ratio was observed (r = −0.422; P < 0.05). Conversely, in 12 acromegalic patients treated by transsphenoidal surgery (GH falling from 124 ± 49.2 to 29.3 ± 15.4 mU/L; P < 0.01), the THF+allo-THF/THE ratio rose from 0.53 ± 0.06 to 0.63 ± 0.07 (P < 0.05). Patients from either group who failed to demonstrate a change in GH levels showed no change in the THF+allo-THF/THE ratio. In vitro studies conducted on cells stably transfected with either the human 11βHSD1 or 11βHSD2 complementary DNA and primary cultures of human omental adipose stromal cells expressing only the 11βHSD1 isozyme indicated a dose-dependent inhibition of 11βHSD1 oxo-reductase activity with IGF-I, but not GH. Neither IGF-I nor GH had any effect on 11βHSD2 activity. GH, through an IGF-I-mediated effect, inhibits 11βHSD1 activity. This reduction in E to F conversion will increase the MCR of F, and care should be taken to monitor the adequacy of function of the hypothalamo-pituitary-adrenal axis in acromegalic subjects and in GH-deficient, hypopituitary patients commencing replacement GH therapy. Conversely, enhanced E to F conversion occurs with a reduction in GH levels; in liver and adipose tissue this would result in increased hepatic glucose output and visceral adiposity, suggesting that part of the phenotype currently attributable to adult GH deficiency may be an indirect consequence of its effect on tissue F metabolism via 11βHSD1 expression.

scholarly journals In Vitro Studies of Synthesis and Release of Factor VIII Related Protein by Endothelial Cells

1977 ◽  
Author(s):  
E. G. D. Tuddenham ◽  
L. W. Hoyer
Keyword(s):  
Endothelial Cells ◽  
Factor Viii ◽  
Calf Serum ◽  
In Vivo Studies ◽  
Separate Experiment ◽  
Related Protein ◽  
Binding Studies ◽  
Radioactive Label

In vivo studies have shown that many stimuli such as epinephrine, exercise and pregnancy lead to a rise in factor VIII levels. However, the physiologic mechanisms controlling factor VIII levels are poorly, if at all, understood. Since endothelial cells synthesize factor VIII related protein (FVIII:RP) and can be grown in tissue culture, they provide a suitable in vitro model to study synthesis and release of FVIII:RP. Endothelial cells were harvested by collagenase digestion from human umbilical cords and grown in medium 199 supplemented with 20 to 30% pooled human serum. Confluent cultures were washed and then maintained in medium 199 supplemented with 20% fetal calf serum. Release of FVIII:RP into the medium was measured by immunoradiometric assay. Labeled amino acids were added to the medium for studies of FVIII:RP synthesis. Incorporation of radioactive label into FVIII:RP was measured in binding studies using a specific immunoadsorbent. Epinephrine in concentrations from 1 ng to 10 ug per ml had no effect on rate of release of FVIII:RP from cultured endothelial cells, suggesting that the in vivo effect of epinephrine is not due to a direct action on endothelial cells. In a separate experiment,exogenous FVIII:RP was added to the culture medium at a high concentration (2 units FVIII:RP per ml) along with 3H Leucine. A control without exogenous VIII:RP incorporated as much radioactivity into VIII:RP as did the culture with added FVIII:RP. This result suggests that there is no end product inhibition of FVIII:RP synthesis which operates on the endothelial cell.

Characterization of the radioiodinated analogue of SCH 23390: In vitro and in vivo D-1 dopamine receptor binding studies

Life Sciences ◽  
1988 ◽  
Vol 43 (14) ◽  
pp. 1151-1160 ◽  
Author(s):  
Robert D. McQuade ◽  
Richard Chipkin ◽  
Nordine Amlaiky ◽  
Marc Caron ◽  
Louis Iorio ◽  
...  
Keyword(s):  
Dopamine Receptor ◽  
Receptor Binding ◽  
Sch 23390 ◽  
Binding Studies ◽  
Dopamine Receptor Binding

scholarly journals Saturation Analysis in PET—Analysis of Errors Due to Imperfect Reference Regions

1994 ◽  
Vol 14 (2) ◽  
pp. 358-361 ◽  
Author(s):  
J.-E. Litton ◽  
H. Hall ◽  
S. Pauli
Keyword(s):  
Receptor Binding ◽  
Specific Binding ◽  
Scatchard Analysis ◽  
Position Emission Tomography ◽  
Reference Region ◽  
Nonspecific Binding ◽  
Binding Studies

In the determination of specific binding in receptor binding techniques in vitro as well as in vivo, determination of the nonspecific binding as well as the free component is of crucial importance. If a low proportion of specific binding is included when determining the nonspecific binding, relatively large errors may be obtained. In the present study, benzodiazepine (BZ) receptor binding in the human brain was determined in vivo using position emission tomography (PET) by applying a saturation procedure using [11C]flumazenil as an example of this problem. Analysis of the errors in Bmax and KD obtained using Scatchard analysis in PET was performed using a priori information from in vitro [3H]flumazenil binding in the pons, used normally as a reference region in BZ receptor binding studies. Even if the density of BZ receptors in the reference region pons is only 2% compared to that in the frontal cortex, this small proportion of specific binding sites will result in a 10% error in the Bmax and KD values. Simulation of a number of Scatchard plots was performed at varying ratios between the nonspecific and the specific binding.

ANDROGEN DEPENDENCE OF RAT PROSTATIC 3α-HYDROXYSTEROID DEHYDROGENASE

1978 ◽  
Vol 79 (1) ◽  
pp. 143-144 ◽  
Author(s):  
R. MASSA ◽  
M. MAS GARCIA ◽  
L. MARTINI
Keyword(s):  
Anterior Pituitary ◽  
Reductase Activity ◽  
Prostate Gland ◽  
Hydroxysteroid Dehydrogenase ◽  
Irreversible Reaction ◽  
Dehydrogenase System ◽  
Rat Prostate ◽  
Biological Actions

Department of Endocrinology, University of Milan, Via A. del Sarto 21, 20129 Milan, Italy (Received 8 May 1978) It is well established that the rat prostate gland converts testosterone mainly into 5α-androstan-17β-ol-3-one (5α-dihydrotestosterone, 5α-DHT) and to a lesser extent into 5α-androstan-3α,17β-diol (5α-tetrahydrotestosterone, 5α-THT). This occurs, both in vivo and in vitro, through the action of a 5α-reductase and a 3α-hydroxysteroid dehydrogenase system (Baulieu, Lasnitzki & Robel, 1968; Bruchovsky & Wilson, 1968; Gloyna & Wilson, 1969; Kniewald, Massa & Martini, 1971). It has also been recognized that, although the 5α-reduction of testosterone is an irreversible reaction, the reduction of 5α-DHT to 5α-THT is reversible (Becker, Grabosch, Hoffmann & Voigt, 1973; Cresti & Massa, 1977). Consequently, the question has been raised as to whether the biological actions of 5α-THT are attributable to the compound as such or to 5α-DHT. At the anterior pituitary level, 5α-reductase activity is increased by castration and decreased

Decreased 5α-dihydrotestosterone catabolism suppresses T lymphocyte functions in males after trauma-hemorrhage

2002 ◽  
Vol 282 (6) ◽  
pp. C1332-C1338 ◽  
Author(s):  
Rui Zheng ◽  
T. S. Anantha Samy ◽  
Christian P. Schneider ◽  
Loring W. Rue ◽  
Kirby I. Bland ◽  
...  
Keyword(s):  
Androgen Receptor ◽  
T Lymphocytes ◽  
Receptor Binding ◽  
T Lymphocyte ◽  
Reductase Activity ◽  
Interleukin 2 ◽  
Hydroxysteroid Dehydrogenase ◽  
Steroidogenic Enzymes ◽  
Intact Male ◽  
High Affinity

Trauma-hemorrhage produces profound immunosuppression in males but not in proestrus females. Prior castration or flutamide treatment of males following trauma-hemorrhage prevents immunosuppression, implicating 5α-dihydrotestosterone for the immunosuppressive effects. 5α-Dihydrotestosterone, a high-affinity androgen receptor-binding steroid, is synthesized in tissues as needed and seldom accumulates. The presence of steroidogenic enzymes in T lymphocytes suggests both synthesis and catabolism of 5α-dihydrotestosterone. We hypothesized, therefore, that the basis for high 5α-dihydrotestosterone activity in T lymphocytes of males following trauma-hemorrhage is due to decreased catabolism. Accordingly, catabolism of 5α-dihydrotestosterone was assessed in splenic T lymphocytes by examining the activity and expression of enzymes involved. Analysis showed increased synthesis and decreased catabolism of 5α-dihydrotestosterone in intact male T lymphocytes following trauma-hemorrhage. In contrast, reduced 5α-reductase activity and increased expression of 17β-hydroxysteroid dehydrogenase oxidative isomers suggest inactivation of 5α-dihydrotestosterone in precastrated males. Thus our study suggests increased synthesis and decreased catabolism of 5α-dihydrotestosterone as a reason for loss of T lymphocyte functions in intact males following trauma-hemorrhage, as evidenced by decreased release of interleukin-2 and -6.

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