Steroidogenesis of the fetal adrenal gland in vitro: influence of metopirone applied in vivo and in vitro.

In vitro conversion of 4-14C-progesterone into corticosteroids in the adrenal glands of rat fetuses treated with Metopirone (Su 4885) on the last day of intrauterine development was studied. After a 1-hr incubation of the adrenal glands of fetuses injected with Metopirone, hydroxylation of progesterone into corticosterone (B), 18-hydroxycorticosterone (18-OH-B) and 18-hydroxy-11-deoxycorticosterone (18-OH-DOC) decreased and the synthesis of 11-deoxycorticosterone increased. Following preincubation of the fetal adrenal glands and 1-hr incubation with Metopirone, hydroxylation of progesterone into DOC increased and the synthesis of B decreased. Preincubation and a 2-hr incubation with Metopirone caused a decrease in the synthesis of B, 18-OH-B and 18-OH-DOC and an increase in DOC. The results constitute direct evidence of the ability of the fetal adrenal glands to synthesize all corticoids and indicate that most probably corticoids are synthesized by the fetal adrenal glands in the same way as in the adrenals of adult animals.     

Serum corticosteroids at the high and low points of the circadian rhythm in rats with regenerating adrenals.

Serum levels of 11-deoxycorticosterone (DOC), 18-hydroxy-11-deoxycorticosterone (18-OH-DOC) and corticosterone (B) were determined at the high (1800 h) and low (0800 h) points of the circadian rhythm in control rats and in rats with regenerating adrenals. The levels of DOC at 0800 h in quiescent rats with regenerating adrenals were 6.5 times greater than in the control group. The levels of 18-OH-DOC and B, however, were not significantly different between these groups. A circadian rhythm for B, 18-OH-DOC and DOC was evident in control rats with a 12,20 and 3.5 fold increase, respectively, at 1800 h as compared to 0800 h. In animals with regenerating adrenals there was only a minimal change in the levels of B and 18-OH-DOC at 1800 h. There was, however, a 2 fold further increase in the levels of DOC at 1800 h as compared with the elevated levels at 0800 h. These findings show that the decrease in 11β and 18-hydroxylase activity of the regenerating adrenal is most clearly evident at the high point of the circadian rhythm. Furthermore, only by taking into account physiological variations in adrenal activity can an accurate assessment of DOC secretion in the adrenal regeneration model of hypertension be obtained.     

Evidence of adrenal 18-hydroxylase inhibition by metyrapone in man.

The influence of metyrapone (M) on the adrenal 18-hydroxylation was studied in two groups of healthy young men. In group I, serum concentrations of 18-OH-11-deoxycorticosterone (18-OH-DOC) fell significantly after a single oral dose of 40 mg/kg of M at 8.00 h, while those of 11-deoxycorticosterone (DOC) increased by a factor of about 500 within 4 hours after drug administration. Serum concentrations of 18-OH-DOC remained suppressed up to 14,00 h and tended to increase up to 16.00 h with a concomitant increase of plasma ACTH. In group II, serum concentrations of 18-OH-DOC and corticosterone (B) were slightly lowered eight hours after oral administration of 30 mg/kg of M at midnight in comparison with measurement of the previous day. Serum concentrations of 11-deoxycortisol (S) and DOC were markedly increased after drug administration. These findings indicate an inhibitory effect of M on adrenal 18-hydroxylation in addition to 11-hydroxylation under in vivo conditions. The slight increase of 18-OH-DOC at 16.00 h in group I and the only slight decrease of this steroid 8 hrs after drug administration in group II may be explained by declining enzyme blockade and a superimposed ACTH stimulation of the adrenal cortex at this time.     

Fetal rat adrenal steroidogenesis and steroid transfer to adrenalectomized mother.

On the 22nd day of gestation in rats, fetuses of acutely adrenalectomized mothers were injected subcutaneously with 0.43 muCi 4-14C-progesterone in 0.05 ml saline. Ten and 20 min after injection to fetuses, samples were taken to determine the 14C-progesterone metabolites in the plasma and adrenal glands. After extraction of the samples taken, the metabolites were separated by two-dimensional thin-layer chromatography and identified by autoradiography. 11-deoxycorticosterone, 18-hydroxy-11-deoxycorticosterone, corticosterone and 11beta-hydroxyprogesterone were identified in the plasma of injected fetuses, and, in far smaller amounts, in the plasma of their mothers. The plasma of noninjected fetuses also contained very small amounts of these corticoids. The fetal adrenal glands contained far smaller amounts of radioactive steroids than the fetal plasma did. The results obtained show that steroids of fetal origin can cross the placenta in and out, constituting evidence that the fetal adrenal glands are the only source of the plasma corticoids of their adrenalectomized mothers.     

An alternative metabolic pathway of 11-deoxycorticosterone in bovine adrenal in vitro: evidence for the presence of a pathway of 11-deoxycorticosterone oxidation at 19-position

Comparative studies of 11 beta-, 18-, and 19-hydroxylation activities of 11-deoxycorticosterone (DOC) by bovine adrenal mitochondria revealed that an appreciable level of hydroxylation rate was observed in 19-hydroxylation (0.32 nmol/min/mg mitochondrial protein), as well as in 11 beta- and 18-hydroxylations (4.7 and 0.27 nmol/min/mg mitochondrial protein, respectively), at saturated substrate concentration in vitro. Also, the rates of the oxidation reactions of 19-hydroxy-11-deoxycorticosterone (19-OH-DOC) and 19-oxo-11-deoxycorticosterone (19-oxo-DOC) at the 19-position were about 5 times higher than the 19-hydroxylation rate of DOC. Although the affinities of 19-OH-DOC and 19-oxo-DOC for the enzyme(s) involved in the C-19 oxidation were about one-fifth those of DOC, these results strongly suggest the presence of the following pathway in bovine adrenal in vitro: DOC----19-OH-DOC----19-oxo-DOC----19-oic-DOC. This was further confirmed by a dynamic study of the formation and subsequent decay of the C-19 oxidized metabolites produced from DOC. At maximum concentrations of 19-OH-DOC and 19-oxo-DOC, the rates of production of, respectively, 19-oxo-DOC and 19-oic-DOC reached maximum. Furthermore, at the beginning of the incubation (1-4 min), an induction period in the formation of 19-oxo-DOC and 19-oic-DOC was observed and the formation of 19-oxo-DOC always preceded the appearance of 19-oic-DOC. These observations strongly support the existence of the pathway of the C-19 oxidation of DOC as mentioned above. It was also established that reduced pyridine nucleotide (NADPH) and molecular oxygen were required for these oxidation reactions. In addition, these three oxidation reactions were uniformly inhibited by the presence of carbon monoxide or metyrapone (0.01-1.0 microM), which is known to bind specifically with cytochrome P-450, while potassium cyanide (0.01-0.1 mM) did not affect them. These results suggest the possibility of the involvement of cytochrome P-450 in the C-19 oxidation reactions of DOC, 19-OH-DOC, and 19-oxo-DOC. We also showed that 19-oic-DOC is not further metabolized to other steroids such as 19-nor-11-deoxycorticosterone in bovine adrenal cortex.     

Stable expression of rat cytochrome P450 11β-Hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2) in MA-10 cells

Glucocorticoids and mineralocorticoids are synthesized in the adrenal cortex through the action of two different cytochrome 11β-hydroxylases, CYP11B1 (11β-hydroxylase) and CYP11B2 (aldosterone synthase) which are distributed in the zona fasciculata and glomerulosa, respectively. We have created stably transfected cell lines using the Leydig tumor cell line MA-10 with CYP11B1 and CYP11B2 cDNA-containing plasmids which have a selectable gene to confer resistance to geneticin. The expression of the transfected cDNA in the cells was characterized by Northern-blot and measurement of enzymatic activity. The cell lines express the enzymes stably for many generations. CYP11B1 transfected cells converted DOC into corticosterone, 18-OH-DOC and small amounts of 18-OH-corticosterone, in a time and concentration dependent manner. Incubation of the cells with corticosterone generated 18-OH-corticosterone especially at concentrations of 30 and 100 μM. The production of 18-OH-corticosterone from corticosterone at these doses was significantly higher than incubations with similar concentrations of DOC. CYP11B2 transfected cells converted DOC into corticosterone, 18-OH-corticosterone, aldosterone and small amounts of 18-OH-DOC in a time and concentration dependent manner. They converted corticosterone into 18-OH-corticosterone and aldosterone in a time and concentration dependent manner. The absolute and relative production of aldosterone from DOC was significantly higher than when cells were incubated with corticosterone, and the ratio of aldosterone to 18-OH-corticosterone was higher at all concentrations of DOC compared to corticosterone. CYP11B2 transfected cells (but not the CYP11B1 transfected cells) transform 18-OH-DOC into 18-OH-corticosterone, but can not convert 18-OH-DOC into aldosterone. In conclusion, stably transfected MA-10 cells with the cDNAs for the CYP11B1 and CYP11B2 enzymes were prepared and their enzymatic activity studied. These cells are useful in the study of inhibitors of the specific enzymes, as well as determining the roles that each enzyme plays in zone-specific steroidogenesis in the adrenal cortex.     

Adaptation of aldosterone biosynthesis to sodium and potassium intake in the rat

The steroidogenic response of rat adrenal zona glomerulosa to stimulators is variable and depends on the activity of biosynthetic steps involved in the conversion of deoxycorticosterone (DOC) to aldosterone (Aldo). Corticosterone methyl oxidations (CMO) 1 and 2 are stimulated by sodium restriction and suppressed by potassium restriction. These slow alterations are accompanied by the appearance or disappearance of a specific zona glomerulosa mitochondrial protein with a molecular weight of 49,000. Induction of CMO 1 and 2 activities and the appearance of the 49 K protein can also be elicited in vitro by culture of rat zone glomerulosa cells in a medium with a high potassium concentration. The 49 K protein crossreacts with a monoclonal antibody raised against purified bovine adrenal cytochrome P-450(11 beta). The same antibody stains a protein with a molecular weight of 51,000 in rat zona fasciculata mitochondria and in zone glomerulosa mitochondria of rats in which CMO 1 and 2 activities have been suppressed by potassium restriction and sodium loading. The 51 K crossreactive protein was purified to electrophoretic homogeneity by chromatography on octyl-sepharose. In a reconstituted enzyme system, it converted DOC to corticosterone (B) and to 18-hydroxy-11-deoxycorticosterone (18-OH-DOC) but not to 18-hydroxycorticosterone (18-OH-B) or Aldo. A partially purified 49 K protein preparation from zona glomerulosa mitochondria of rats kept on a low-sodium, high-potassium regimen converted DOC to B, 18-OH-DOC, 18-OH-B and Aldo. According to these results, rat adrenal cytochrome P-450(11 beta) exists in two different forms, with both of them capable of hydroxylating DOC in either the 11 beta- of the 18-position, but with only the 49 K form capable of catalyzing CMO 1 and 2. The adaptation of aldosterone biosynthesis to sodium deficiency or potassium intake in rats is due to the appearance of the 49 K form of the enzyme in zona glomerulosa mitochondria.     

ACTH and growth hormone relationship in fetal rat adrenal steroidogenesis in vitro.

The effects of growth hormone and ACTH, alone or in combination, on fetal rat adrenal steroidogenesis in vitro were examined on the last day of intrauterine development. ACTH increased, while growth hormone did not affect fetal adrenal weight. ACTH increased fetal rat adrenal steroidogenesis, hydroxylation of 4-14C-progesterone to corticosterone, 18-hydroxy-11-deoxycorticosterone, 11-hydroxycorticosterone and aldosterone. Growth hormone alone had no effect on fetal adrenal steroidogenesis. ACTH and growth hormone administered together increased the conversion of progesterone to the above mentioned steroids to a greater extent than ACTH alone. The results indicate that growth hormone may participate in the fetal rat adrenal steroidogenesis potentiating the effects of fetal pituitary ACTH.     

Inhibition of aldosterone production in rat adrenal mitochondria by 18-ethynyl-11-deoxycorticosterone: a simple model for kinetic interpretation of mechanism-based inhibitors

A simple mathematical model for studying mechanism-based inhibitors (MBIs) is presented. The mathematical equations are deduced for an experimental protocol consisting of a first incubation of the enzyme in the presence of MBI followed by a washing protocol to eliminate free MBI. Finally enzyme activity (initial velocity) is measured with specific substrate. The representation of the final equation obtained is a straight line, and the MBI-specific association constant of velocity (k) can be calculated from its slope. The mathematical model was then challenged with the effect of 18-ethynyl-11-deoxycorticosterone (18-EtDOC) as an MBI on aldosterone biosynthesis from 11-deoxycorticosterone (DOC) in rat adrenal mitochondria. The last step of the mitochondrial biosynthesis of aldosterone consists of the conversion of DOC into corticosterone (B) or 18-hydroxy-11-deoxycorticosterone (18-OHDOC), and both steroids can then be transformed into aldosterone. The k (mM(-1) x min(-1)) values obtained for 18-EtDOC were: 451 +/- 36 for DOC to aldosterone; 177 +/- 16 for B to aldosterone; 175 +/- 15 for 18-OHDOC to aldosterone; and 2.7 +/- 0.2 for DOC to B. These results show that this MBI practically does not affect the metabolism of DOC to B in our enzyme preparation and that conversions of B and 18-OHDOC into aldosterone are catalyzed by the same enzyme.     

Activation of adrenal function in fetal sheep by the infusion of adrenocorticotropin to the fetus in utero

We determined whether ACTH1-24, infused into fetal lambs at a rate that is known to cause premature labor, elicits changes in the responsiveness of the fetal adrenal glands, and alters the pattern of corticosteroid output. Plasma cortisol (F), corticosterone (B) and progesterone (P4) were measured during 72 h of infusion of saline or ACTH (10 micrograms/h) beginning on Day 127 of pregnancy. Adrenals were then dispersed into isolated cells, and the output of F, B and P4 after exogenous ACTH determined in vitro. Plasma concentrations of F and B were higher in ACTH-treated fetuses. The increment in F (5-to 7-fold) was greater than that in B (2-fold) such that the F:B ratio in plasma of ACTH-treated fetuses on Days 2 and 3 of infusion was 2.5 times higher than in controls. After 72 h of infusion, the adrenal weights in ACTH-treated fetuses (741 +/- 38 mg, +/- SEM; n = 4) were greater than in the control animals (349 +/- 11 mg). There was a significant effect of ACTH pretreatment in vivo on F output by isolated adrenal cells in vitro. Mean increments in F output after addition of ACTH1-24 (5000 pg/ml) in vitro rose from 368 +/- 235 pg/50,000 cells in controls, to 64,639 +/- 19,875 pg/50,000 cells after ACTH in vivo. There was no significant effect of ACTH in vivo on B output in vitro; the ratio of F:B output, either in the absence or presence of ACTH in vitro, was significantly higher in cells from ACTH-pretreated fetuses. There was a significant effect of in vivo ACTH on in vitro P4 output. After ACTH treatment in vivo there was an increase in the vitro output ratio of F:P4, but no change in the output ratio of B:P4. We conclude that ACTH treatment of the fetal lamb in vivo results in activation of fetal adrenal function, increased fetal adrenal responsiveness to ACTH, and directed corticosteroid biosynthesis towards cortisol. Our results are consistent with an increase in fetal adrenal 17 alpha-hydroxylase activity after ACTH treatment.     

Androstenedione-mediated inhibition of 11 beta-hydroxylation in monolayer cell cultures of fetal calf adrenals

The possibility that the formation of androstenedione by fetal calf adrenal cells in culture is linked to their decreased ability to form cortisol and corticosterone was investigated. Fetal calf adrenal cells metabolise radioactive adrostenedione to two major products which coelute on thin layer chromatography with 11 beta-hydroxyandrostenedione and 11 beta-hydroxytestosterone. When the cells are incubated with 11-deoxycortisol or 11-deoxycorticosterone in the presence of androstenedione there is a dose dependant inhibition of cortisol and corticosterone formation. Further studies with progesterone showed an accumulation of 11-deoxycortisol and 11-deoxycorticosterone in cells incubated simultaneously with androstenedione. The results demonstrate that exogenous androstenedione can have dramatic effects on steroidogenesis in the fetal calf adrenal and suggest that the accumulation of androstenedione in the medium of cultured andrenocortical cells is responsible, at least in part, for the decreased formation of cortisol and corticosterone.     

Altered secretion of corticosteroids and prolactin in adrenal regeneration hypertensive rats.

To assess the possible role of mineralocorticoids in the onset and maintenance of hypertension in adrenal regeneration hypertensive (ARH) rats, the change in plasma mineralocorticoids, with adrenal regeneration after enucleation in ARH rats was investigated and compared with those in unilaterally nephroadrenalectomized, 1% saline-fed (UNA) rats, sham-operated, 1% saline-fed (1% NaCl) rats and water-fed (water) rats. Plasma aldosterone was determined by RIA and the other mineralocorticoids were measured by HPLC. How plasma PRL, a marker of central dopaminergic activity, affected aldosterone secretion was determined by RIA. In ARH, plasma corticosterone (B), 18-OH-DOC and aldosterone levels 2 weeks after operation were as low as 20-30% of corresponding values, but the plasma DOC level was almost 100% of the corresponding value in the other groups. Four weeks after operation plasma B increased to a level comparable with that in the other groups and the plasma aldosterone level remained low. However, plasma DOC and 18-OH-DOC levels 4 weeks after operation were as high as 120-200% of corresponding values in the other groups. Six weeks after operation, the plasma aldosterone level returned to a value comparable with that in UNA and 1% NaCl and plasma DOC and 18-OH-DOC levels returned to corresponding values in the other groups. The plasma PRL level 4 weeks after operation was significantly lower in ARH than in the other groups. These results suggest that transient DOC and 18-OH-DOC increases observed in ARH may be important in the onset of hypertension, while other factors may be involved in its maintenance and that the transient central dopaminergic hyperactivity observed in ARH may be responsible for a delayed return from aldosterone deficiency.     

Plasma levels of 18-hydroxy-11-deoxycorticosterone in essential hypertension.

In order to investigate the role of 18-hydroxy-11-deoxycorticosterone (18-OH-DOC) in essential hypertension (EH), the responses of plasma 17-OH-DOC to 7 stimulation tests (furosemide test, adrenal suppression test, angiotensin II infusion test, adrenal stimulation test, metopirone test, saline infusion test and potassium chloride infusion test) and the circadian rhythm were investigated in 18 patients with essential hypertension (low renin group: 8, and normal renin group: 10). From the present study, it micht be thought that plasma 18-OH-DOC does not play an important role in the suppression of PRA in patients with low PRA.     

An adrenocortical tumor secreting weak mineralocorticoids

An adrenocortical carcinoma (15.5 g) secreting excessive amounts of steroids with weak mineralocorticoid activity in a 25-year-old woman was studied with particular reference to its in vivo and in vitro secretions of steroids. Severe hypertension, occasional low serum potassium and suppressed PRA were the major clinical findings, and were improved with removal of the tumor. In the preoperative stage, plasma levels of 11-deoxycorticosterone, 18-hydroxy-11-deoxycorticosterone, corticosterone and 18-hydroxycorticosterone were all increased. However, the plasma level of aldosterone was repeatedly normal. Although plasma levels of pregnenolone, 17-hydroxypregnenolone, progesterone and 17-hydroxyprogesterone were very high, those of other late step steroids, i.e. 11-deoxycortisol, cortisol, dehydroepiandrosterone, androstenedione and testosterone were almost normal. From these findings, a major etiological role of weak mineralocorticoids such as 11-deoxycorticosterone, 18-hydroxycorticosterone and corticosterone in her hypertension was suggested. Pregnenolone and 17-hydroxypregnenolone in tumor tissue were increased, but 11-deoxycorticosterone, corticosterone, aldosterone, cortisol and adrenal androgens such as dehydroepiandrosterone, androstenedione and testosterone were below normal or low normal. In vitro production of 11-deoxycorticosterone, aldosterone or cortisol by the tumor tissue slices was very low and scarcely responded to synthetic ACTH.     

Biotransformation of pregnenolone - 7alpha-3H, progesterone - 7alpha-3H and dehydroepiandrosterone - 7alpha-3H by porcine fetal and maternal adrenal homogenate preparations.

The biotransformation of pregnenolone-7alpha-3H and of progesterone-7alpha-3H by porcine fetal and maternal adrenal homogenates at 56 and 112 days of pregnancy and of dehydroepiandrosterone-7alpha-3H by fetal adrenal homogenates has been investigated in vitro. Both pregnenolone-7alpha-3H and progesterone-7alpha-3H were metabolized extensively by maternal adrenal preparations, the principal radioactive metabolites isolated being cortisol, corticosterone, 11-deoxycortisol, deoxycorticosterone, 11beta-hydroxyprogesterone and androstenedione. In addition, 17alpha-hydroxyprogesterone, 20alpha-dihydroprogesterone and cortisone were formed from both substrates and 17alpha-hydroxypregnenolone and progesterone were formed from pregnenolone. Although essentially the same radioactive metabolites were isolated after incubation of fetal adrenal glands with pregnenolone-7alpha-3H or progesterone-7alpha-3H, a greater proportion of the radioactivity was associated with corticosteroids at 112 days of pregnancy than at 56 days. 11beta-Hydroxyandrostenedione and androstenedione were isolated and identified together with an unknown polar metabolite, after incubation of fetal adrenal tissue with dehydroepiandrosterone-7alpha-3H. These results are discussed in relation to feto-placental steroid biosynthesis and metabolism and the role of the fetal adrenal in the initiation of parturition in the pig.     

Conversion of 11-deoxycorticosterone and corticosterone to aldosterone by cytochrome P-450 11 beta-/18-hydroxylase from porcine adrenal

Highly purified cytochrome P-450 11 beta-/18-hydroxylase and the electron carriers adrenodoxin and adrenodoxin reductase were prepared from porcine adrenal. When the enzyme was incubated with the electron carriers, 11-deoxycorticosterone (DOC) and NADPH, the following products were isolated and measured by HPLC: corticosterone, 18-hydroxy-11-deoxycorticosterone (18-hydroxyDOC), 18-hydroxycorticosterone and aldosterone. All of the DOC consumed by the enzyme can be accounted for by the formation of these four steroids. Aldosterone was identified by mass spectroscopy and by preparing [3H]aldosterone from [3H]corticosterone followed by recrystallization at constant specific activity after addition of authentic aldosterone. Corticosterone and 18-hydroxycorticosterone were also converted to aldosterone. Conversion of corticosterone and 18-hydroxycorticosterone to aldosterone required P-450, both electron carriers, NADPH and substrate. The reaction is inhibited by CO and metyrapone. Moreover, all three activities of the purified enzyme decline at the same rate when the enzyme is kept at room temperature for various periods of time and when the enzyme is treated with increasing concentrations of anti-11 beta-hydroxylase (IgG) before assay. It is concluded that cytochrome P-450 11 beta-/18-hydroxylase can convert DOC to aldosterone via corticosterone and 18-hydroxycorticosterone. The stoichiometry of this conversion was found to be 3 moles of NADPH, 3 moles of H+ and 3 moles of oxygen per mole of aldosterone produced.     

18-Hydroxy-11-deoxycorticosterone in hypertensive uremic patients during hemodialysis.

The present study was carried out in 25 hypertensive uremic patients on regular 4 h dialysis, 3 times a week. Plasma 18-hydroxy-11-deoxycorticosterone (18-OH-DOC), aldosterone (PA) and corticosteroids were determined by radioimmunoassay and competitive protein binding technique before and at the end of the 1st, 2nd and 3rd hour of hemodialysis. Plasma 18-HD-DOC was normal before dialysis and did not change significantly during hemodialysis, whereas body fluids and electolytes decreased progressively. No correlation was observed between blood pressure and 18-OH-DOC during dialysis. 18-OH-DOC did not correlate with PA which decreases progressively during hemodialysis and was correlated to plasma corticosteroids only at the 3rd hour of dialysis, probably on account of the enhanced influence of ACTH on the adrenal cortex.     

Localization of aldosterone and corticosterone in the central nervous system,assessed by quantitative autoradiography

Nuclear localization of tritiated aldosterone in the CNS was studied in rats by numerical evaluation of silver grains, deposited over neuronal cell nuclei in thawmounted autoradiograms, and compared with the localization obtained after prior administration of a 100-fold excess of radioinert aldosterone, corticosterone or 18-hydroxy-11-deoxycorticosterone (18-OH-DOC). Corticosterone and 18-OH_DOC completely prevented nuclear localization in most regions examined. However, in contrast to pretreatment with aldosterone, pretreatment with corticosterone and 18-OH-DOC did not completely prevent the concentration of radio-activity in the cell nuclei of the indusium griseum. Traces of radioactivity were, furthermore, retained in areas CA₁ and CA₂ and the dentate gyrus in rats exposed to corticosterone, but not to 18-OH-DOC, prior to [³H]aldosterone. A similar profile of silver grain distribution to that noted with aldosterone was found for corticosterone except that with tritiated corticosterone the most intense concentration of radioactivity occurred in hippocampal areas CA₁ and CA₂ and not in the indusium griseum. Prior administration of excess deoxycorticosterone acetate abolished nuclear accumulation of tritiated corticosterone. Dihydrotestosterone, on the other hand, failed to compete with tritiated corticosterone at a dose 200-fold in excess of the tritiated steroid.We conclude that (1) a receptor readily shared by aldosterone, corticosterone, 18-OH-DOC and DOC, but not by dihydrotestosterone, is widely distributed throughout the CNS, (2) a receptor shared by aldosterone and 18-OH-DOC, but not by corticosterone may be present in hippocampal areas CA₁ and CA₂, (3) that both these as well as the receptor accepting dihydrotestosterone can be located within the same cell.Dedicated to K. A. C. Elliott on his 80th birthday.     

The role of ACTH in determining the metabolic pathways of deoxycorticosterone by newborn rat adrenal cells in primary culture

The metabolism of deoxycorticosterone (DOC) by newborn rat adrenal cells in primary culture at various times after culture, with and without ACTH, was studied. After 5 days in culture before addition of ACTH, the main products of the metabolism of DOC were corticosterone and 18-hydroxy-11-deoxycorticosterone in a 2:1 ratio. Smaller amounts of 20 alpha-dihydrocorticosterone and 18-hydroxycorticosterone were also found. No reduced metabolites of DOC were detected. Without ACTH the conversion of DOC to corticosterone and 18-hydroxyDOC declined rapidly. After 13 days in culture, this conversion accounted for only half the metabolites. The reductive metabolism of DOC which yields products reduced at 20 alpha and/or 3 alpha/beta and 5 alpha accounted for the other half. When ACTH (22 mU/ml) was added to the culture daily for several weeks, the primary metabolism of DOC remained that of 11 beta- and 18-hydroxylation yielding corticosterone and 18-hydroxyDOC. A minor reductive metabolism was found. Both cultures produced 6 beta-hydroxyDOC. These results demonstrate that ACTH is needed to maintain the efficiency of the 11 beta/18-hydroxylating system. They also show that ACTH controls the type of metabolism predominant in the rat adrenal cell and may be responsible for the balance between the biosynthesis of glucocorticoids and their reductive catabolism in the fasciculata zone of the adrenal gland.     

In vivo and in vitro studies on steroid metabolism in a case of primary aldosteronism with multiple lesions of adenoma and nodular hyperplasia

In order to systematically analyze the regulation and metabolism of steroid hormones in a case of primary aldosteronism with multiple lesions, including adenoma and nodular hyperplasia of the left adrenal gland, the amounts of 9 steroids (progesterone (P), 11-deoxycorticosterone (DOC), corticosterone (B), 18-hydroxycorticosterone (18-OH-B), aldosterone (Aldo), 17 alpha-hydroxyprogesterone (17-OH-P), 11-deoxycortisol (S), cortisol (F) and dehydroepiandrosterone sulfate (DHEAS)) contained in the plasma and in the adrenal tissues were measured. The patient (a 39-year-old female) was admitted to our hospital because of hypokalemia and hypertension. A diagnosis of primary aldosteronism was made on the basis of a complete evaluation, and an adenoma (1.8 x 1.2 cm), a nodular hyperplasia (0.5 x 0.5 cm), a microadenoma and a cortical nodule were found on the left adrenal gland. In vivo studies revealed that the plasma level of Aldo was high, but those of the other steroid hormones were within the normal range. After ACTH infusion, the plasma levels of the 9 steroid hormones increased by 2 to 17 times the base levels. In particular, the responses of DOC and B were markedly high. In vitro studies on P, DOC, B, Aldo and F content in the adenoma (A), the nodular hyperplasia (A'), the adjacent adrenal tissue (C) and the right normal adrenal tissue (D) revealed that, except for F, they were highest in A, followed by A', D and C in that order. In incubation studies with ACTH using A and C, it was found that the levels of 8 steroid hormones with the exception of DHEAS were high in A than in C. In particular, the response of B in A was markedly increased. These findings suggest that aldosteronoma produces 8 steroid hormones under conditions of excess ACTH, while at physiological levels of ACTH, it produces only Aldo in excess.