Evidence of 11 beta-hydroxylase deficiency in a patient with cortical adrenal adenoma.

We explored a 61 year old woman with mild hirsutism. An adrenal tumor was found in the left adrenal, which was held responsible for the androgen secretion. The in vitro incubation of the tumor tissue showed an impaired 11 beta-hydroxylation of 11-deoxycortisol. This is a rare and unusual case of adrenal pathology showing that a deficiency in 11 beta-hydroxylase activity does not rule out the presence of an adrenocortical adenoma.

Effect of long term deprivation of luteinizing hormone on Leydig cell volume, Leydig cell number, and steroidogenic capacity of the rat testis.

Leydig cells atrophy, losing cytoplasmic volume and the capacity for testosterone secretion, within 1-2 weeks of LH deprivation. We investigated the effects of long term (0-16 weeks) LH deprivation on the volume of an average Leydig cell, the volume of Leydig cells per testis, the number of Leydig cells per testis, and testosterone secretion by in vitro perfused testes. Endogenous LH was suppressed in adult rats by testosterone/estradiol-filled (TE) Silastic implants. The presence of Leydig cells in testes was verified by 1) morphological examination using light and electron microscopy, 2) histochemical localization of 3 beta-hydroxysteroid dehydrogenase activity (3 beta HSD), and 3) conversion of pregnenolone to progesterone by in vitro perfused testes. Marked quantitative differences existed in Leydig cell morphology among control and treated rats. The volume of an average Leydig cell and the total volume of Leydig cells per testis decreased (P less than 0.01) rapidly and progressively after TE implantation. At 16 weeks, the average Leydig cell lost 90% of its cytoplasmic volume and 65% of its nuclear volume. Analysis of variance failed to detect a significant decline in Leydig cell number per testis, despite a 16% reduction from the value in control rats (22.2 +/- 1.5 x 10(6)) in rats treated for 16 weeks (18.7 +/- 1.5 x 10(6)). After TE implantation, LH-stimulated testosterone secretion by in vitro perfused testes diminished (P less than 0.01) rapidly to 5% of the control values at 1 week and less than 0.3% of the control value from 4-16 weeks. In contrast, 25% of 3 beta HSD activity was retained (P less than 0.01 vs. controls) at 16 weeks, based on the rate of pregnenolone conversion to progesterone. Moreover, testes of treated rats secreted progesterone at a rate twice that of controls, when the steroid secretion rates were expressed per volume of Leydig cell cytoplasm. Loss of the testosterone-secreting capacity of testes after LH withdrawal was associated with a loss in the volume, but not a significant loss in the number, of Leydig cells. Thus, LH was required to maintain the differentiated structure and function of Leydig cells, but was not required to maintain the overwhelming majority of Leydig cells in the adult rat testis through 16 weeks. Moreover, at least one steroidogenic enzyme, 3 beta HSD, was retained by Leydig cells after long term LH deprivation.

Factors involved in interferon-induced or cholera toxin-induced steroidogenesis in Y-1 mouse adrenal tumour cells.

In addition to its antiviral effect, interferon, at high concentrations, stimulates steroidogenesis and provokes cell rounding in Y-1 mouse adrenal tumour cells. This stimulation was inhibited by cytochalasin B and colchicine. In contrast, dibutyryl cAMP and cholera toxin, also able to induce steroid production and cell rounding, increased steroid production even in the presence of these cytoskeleton-disrupting agents. The initial trigger for interferon or cholera toxin thus probably involves a distinct receptor organization. However, since both inducers increased cAMP synthesis in this differentiated cell line, the further metabolic steps of ketosteroid production could be the same.

11 beta-Hydroxy-11-ketosteroids equilibrium, a source of misinterpretation in steroid synthesis: evidence through the effects of trilostane on 11 beta-hydroxysteroid dehydrogenase in sheep and human adrenals in vitro.

Trilostane is known as an inhibitor of 3 beta-hydroxysteroid dehydrogenase. Conflicting data published on this drug led us to look for the effects of 0.02 to 0.5 mM of trilostane on the in vitro steroid synthesis in sheep adrenals and human adrenals (Cushing's or Conn's syndrome) in the presence of an NADPH-generating system. The synthesis of 4-androstenedione, 11 beta-hydroxyandrostenedione and 11-ketoandrostenedione were studied either from dehydroepiandrosterone or 4-androstenedione or 11 beta-hydroxyandrostenedione. The synthesis of 11-deoxycortisol, cortisol, cortisone, 4-androstenedione, 11 beta-hydroxyandrostenedione and 11-ketoandrostenedione were studied either from 17-hydroxyprogesterone or 11-deoxycortisol or cortisol. This study showed that trilostane inhibited 3 beta-hydroxysteroid dehydrogenase whereas it had no effect on 21-, 11- and 17-hydroxylase. Trilostane was responsible for an increased 11 beta-hydroxysteroid dehydrogenase activity in vitro, resulting in low yields of cortisol and 11 beta-hydroxyandrostenedione, and high yields of cortisone and 11-ketoandrostenedione. This unexpected effect of trilostane allowed us to show that erroneous conclusions (in this case: pseudo inhibition of 11 beta-hydroxylase) can be drawn if all the metabolic pathways from a determined precursor are not exhaustively documented when studying the effects of drugs on steroid synthesis in vitro. The decrease of cortisol synthesis by trilostane may thus be related to the effects of the drug on both 3 beta-hydroxysteroid-dehydrogenase (inhibitory effect) and 11 beta-hydroxysteroid-dehydrogenase (stimulatory effect). This latter effect was found to be species-dependent.

Formation and isolation of delta 5-3-ketosteroids using a purified rat liver alcohol dehydrogenase.

3 beta-Hydroxy-5-androsten-17-one is converted to 5-androstene-3, 17-dione by rat liver alcohol dehydrogenase (ADH). We have reported on the purity of the enzyme which is eluted with pyrazole as a single homogeneous protein using an AMP-agarose affinity column. Rat liver ADH can oxidize hydroxyl groups not only at 3 beta-, but also at 3 alpha-, and 17 beta-positions to a lesser extent; thus it is a pure mammalian enzyme with multifunctional activity for steroids. Since it does not contain delta 5-isomerase activity, the reaction of the dehydrogenase to form the delta 5-ketosteroid intermediate can be observed at pH 7.0, 25 degrees C. Similarly, intermediary product, 5-pregnene-3,20-dione, can be isolated in the conversion of pregnenolone by ADH to progesterone. With buffer alone in a cuvette, a non-enzymatic isomerization of the delta 5-3-ketone occurs at a slow rate (t 1/2 = 6 hrs) but occurs rapidly during isolation procedures. The delta 5-3-ketosteroid intermediates were identified by their behavior on TLC plates with UV light and by their characteristic spectra in the NMR.

The control of steroidogenesis by human fetal adrenal cells in tissue culture. II. Comparison of morphology and steroid production in cells of the fetal and definitive zones.

Preparations of dispersed human fetal adrenal cells from the inner third of the gland and from the subcapsular area were maintained in culture, and their ultrastructure and steroid production were studied. The former type of preparation contained only fetal zone cells, while the latter contained definitive zone cells together with varying numbers of fetal zone cells. Both types could be cultured with equal ease, but during short term culture, fetal and definitive zone cells became morphologically indistinguishable. The patterns of steroid production and, in particular, the relative production of delta 4,3-ketosteroids and delta 5,3 beta-hydroxysteroids were similar in both preparations, as were their dose-response relationships during incubation with alpha ACTH-(1-24). Although considerable variability in total steroid production was observed between cells from different adrenal glands, in no specimen was any evidence for functional zonation of the fetal adrenal cortex observed in vitro. The results suggest that the apparently unique histological appearance and function of the fetal adrenal cortex may only reflect intense stimulation by ACTH secondary to the combined influences of a rapid cortisol MCR and of some inhibitor of fetal adrenal 3 beta-hydroxysteroid dehydrogenase activity.

Induction of delta 3(4) ketosteroid synthesis by interferon in mouse adrenal tumour cell cultures.

Interferon, like cholera toxin, induces in mouse adrenal YI cells the production of delta 3(4) ketosteroids. In parallel, cyclic AMP (cAMP) is activated. At high concentrations, mouse but not human interferon induces a cell rounding effect, similar to that obtained with cholera toxin. In YI cells transformed by simian adenovirus 7, production of cAMP is still increased by that of steroids is blocked. The role if interferon in these cellular events is discussed.

Morphological and steroidogenic changes in cultured adrenal tumor cells induced by a subunit of cholera enterotoxin.

A purified subunit of the cholera enterotoxin molecule was found to have morphological and steroidogenic inducing effects similar to those induced by the native enterotoxin on monolayer tissue cultures of Y1 adrenal tumor cells, although 1,000 times more subunit than toxin (weight basis) was required for maximal effects. In contrast to the whole toxin, the effects of the active subunit could not be prevented by prior incubation with either Gm1 ganglioside or with antibodies directed against choleragenoid (the binding subunit). These results suggest that different receptor sites may exist on cells for the binding and for the active subunits of cholera enterotoxin and/or that the active toxin fragment may exert its effects after gaining access to the intracellular compartment.

Formation of 5alpha-reduced C19-steroids from progesterone in vitro by a pathway through 5alpha-reduced C21-steroids in ovaries of late prepubertal rats.

Ovarian homogenates from 10-150-day-old rats were incubated with [3H]progesterone and NADPH. Also, ovarian homogenates from 28-day-old rats were incubated for 5-180 min with either [14C]progesterone, [3H]5alpha-pregnane-3,20-dione or [14C]progesterone plus [3H]5alpha-pregnane-3,20-dione. Following incubation, radioactive metabolites were isolated, identified, and measured by column and paper chromatography, with derivative formation and recrystallizations to constant specific activity. Prepubertal ovaries (10, 20, and 28 days of age) converted 15-60% of progesterone to C21-17-hydroxysteroids and C19-steroids. At 40 and 150 days of age (postpubertal), the formation of these steroids decreased to less than 2%. At 10 and 150 days of age, the major C19-steroids formed from progesterone were androstenedione and testosterone. At 20 and 28 days of age, however, no accumulation of these C19-delta4-3ketosteroids was found (less than 0.1% of each), at which time the conversion of progesterone to 5alpha-reduced C19-steriods, such as androsterone and 5alpha-androstane-3alpha,17beta-diol, reached 30%. In ovaries of 28-day-old rats, the results from incubation studies for the detection of metabolic pathways indicated two biosynthetic pathways leading to 5alpha-reduced C19-steroids, one from progesterone via 5alpha-reduced C21 steroids, such as 3alpha-hydroxy-5alpha-pregnan-20-one and 3alpha,17alpha-dihydroxy-5alpha-pregnan-20-one, and a second via 17-hydroxyprogesterone, androstenedione, and testosterone. It seems that the active 5alpha-reduction of C19-delta4-3-ketosteroids and the formation of 5alpha-reduced C19-steroids by the pathway through 5alpha-reduced C21-steroids, are present in the ovaries of older prepubertal rats and may be the biological significance.

Hormonal control of Luteal 20alpha-Hydroxy steroid dehydrogenase and delta5-3beta-hydroxy steroid dehydrogenase during luteolysis in the pregnant rat.

Treatment of pregnant rats with human chorionic gonadotrophin, luteotrophin (luteinizing hormone), luteotrophin-releasing hormone, prostaglandin F2alpha, aminoglutethimide, or by foetoplacental removal or hysterectomy achieved a common multiple-response pattern, namely increased activity of luteal 20alpha-hydroxy steroid dehydrogenase with decreased activity of delta5-3beta-hydroxy steriod dehydrogenase and release of delta4-3-oxo steroids in vitro. 2. Similar effects of foetoplacental removal are noted in pregnant mice. 3. Gonadotrophin induced lower activities of 20alpha-hydroxy steroid dehydrogenase, except at the very end of pregnancy, and partly inhibited the induction caused by foetoplacental removal. 4. The results suggest that existence of a placental factor that restrains these changes until the end of normal pregnancy, which is produced in amounts proportional to the number of placentae and is conveyed to the ovary via the blood. 5. This factor was not replaced by prolactin. 6. It is argued that neither placental lactogen nor pituitary luteotrophin participate in the induction of 20alpha-hydroxy steroid dehydrogenase at late pregnancy in the rat. 7. Aminoglutethimide induced 20alpha-hydroxy steroid dehydrogenase only in late pregnancy. This was partly reversed by progesterone, wholly reversed by progesterone plus oestrogen, and did not involve the pituitary.

Luteal 20alpha-hydroxy steroid dehydrogenase and the formation of delta4-3-oxo steroids in the rat after weaning or treatment with 2-bromo-alpha-ergocryptine during lactation.

Natural or early weaning of rat litters caused an increased activity of maternal luteal 20alpha-hydroxy steroid dehydrogenase and a decreased release of delta4-3-oxo steroids in vitro. 2. Compound CB-154 (2-bromo-alpha-ergocryptine) caused an increase of 20alpha-hydroxy steroid dehydrogenase activity in mid-lactation but not in early lactation. 3. Prolaction did not prevent these increases in enzyme activity.

PIP: Luteal 20alpha-hydroxysteroid dehydrogenase and the formation of delta-4-3-oxo steroids after weaning, and treatment with 2-bromo-alpha-ergocryptine during lactation were investigated in the rat. Maternal luteal 20alpha-hydroxy steroid dehydrogenase activity on Day 10 of lactation was greatly increased when the litter had been removed 0-3 days before. In contrast, a decrease in the release of delta-4-3 oxo steroids in vitro was seen with early removal of the litter. The increase in 20alpha-hydroxy steroid dehydrogenase activity due to litter removal was not prevented by the simultaneous injection of prolactin. 2-broma-alpha-ergocryptine caused an increase of 20alpha-hydroxysteroid dehydrogenase activity in midlactation but not in early lactation. Prolactin did not prevent the increase. It is suggested that luteotrophin could possibly account for the enzyme induction after weaning.

Classification of enterotoxins on the basis of activity in cell culture.

Two cell culture systems were used in a study of the biological properties of several bacterial enterotoxins in vitro. By means of one model, in which HeLa cell monolayers were used, cytotoxic effects, interms of detachment of cells from a glass surface due to cell death, were assayed. By means of the second model, activation of the adenyl cyclase-cyclic adenosine 3', 5'-monophosphate (AMP) system, in terms of increased steroidogenesis by Y-1 adrenal cells (an effect which we have termed cytotonic), was assayed. None of the four toxins manifested both properties. Rather there was a clear segregation into two cytotoxic enterotoxins (Shigella dysenteriae type 1 and Clostridium perfingens) and two cytotonic products (Vibrio cholerae and Escherichia coli). These data raise the possibility that some enterotoxins may not interact with the adenyl cyclase-cyclic AMP system; this possibility has also been suggested by studies of the toxin of S. dysenteriae type 1 in the rabbit small bowel. These cell culture systems may therefore serve as convenient models for the study of the mechanism of action of both classes of enterotoxin.