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.     

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.     

Influence of ACTH secreting tumor (MtTF4) on fetal rat adrenal gland steroidogenesis in vitro in prolonged pregnancy

Pregnant female rats with ACTH secreting tumor (MtTF4) have prolonged pregnancy and cannot deliver. The fetuses of tumor bearing females have in prolonged pregnancy on days 24 and 25 of pregnancy greater body weight and smaller adrenal weight as compared to intact fetuses of the 22nd day of pregnancy. The fetal adrenal glands converted to vitro 4-14C progesterone to radioactive 11-deoxycorticosterone (DOC), corticosterone (B), 18-hydroxy-11-deoxycorticosterone (18-OH-DOC), 18-hydroxy-corticosterone (18-OH-B) and aldosterone. Fetal adrenal glands in prolonged pregnancy synthetized in vitro less amount of radioactive DOC, B and 18-OH-DOC. A negative relationship exists between the maternal corticosterone which passes the placenta to fetuses and corticosteroidogenesis of fetal adrenal glands. These results indicate the possibility that fetal rat adrenal glands with their corticosteroids participate in pregnancy and influence normal delivery.     

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.     

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.     

19-Hydroxylation of 18-hydroxy-11-deoxycorticosterone by adrenal mitochondria prepared from various animal species

We have recently reported that bovine adrenocortical cytochrome P-45011 beta catalyzes 19-hydroxylation of 18-hydroxy-11-deoxycorticosterone (18(OH)DOC) in addition to 11 beta-hydroxylation of the steroid. In this report, we examine the presence of these two activities in 18(OH)DOC and 11 beta- and 18-hydroxylation activities on deoxycorticosterone (DOC) among the adrenal mitochondria prepared from man, ox, pig, rabbit, guinea-pig and rat. The results indicate that these animals could be classified into three groups with respect of these hydroxylation activities. Mitochondria of the first group comprising ox and pig showed rather high 19- and 11 beta-hydroxylation activities on 18(OH)DOC compared to the hydroxylation activities on DOC. Mitochondria prepared from the second group which comprised rabbit, guinea-pig and man showed low 19-hydroxylation activity on 18(OH)DOC, whereas the 11 beta-hydroxylation of 18(OH)DOC well occurred in these species. The last group comprising rat had very low activity both of 11 beta- and 19-hydroxylations when 18(OH)DOC was used as the substrate, whereas both 11 beta- and 18-hydroxylations of DOC were high in rat adrenal mitochondria. No significant difference of these activities could be found between zona glomerulosa cells and zonae fasciculata-reticularis cells of bovine adrenal cortex, and between adrenal mitochondria from spontaneously hypertensive rat and those from WKY normotensive rat.     

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.     

Effects of ACTH on plasma levels of adrenal steroids in essential hypertension.

Plasma concentrations of progesterone (P), deoxycorticosterone (DOC), 17-hydroxyprogesterone (17-OH P), corticosterone (B), deoxycortisol (S), cortisol (F) and aldosterone (A) in 8 control subjects (mean age: 40.5 years) and 10 patients with essential hypertension (EH) (mean age: 48.5 years) were determined before, 4 and 8 hours after an infusion of ACTH at a rate of 25 units per 8 hours. Secretion rates (SR) of 18-hydroxy-11-deoxycorticosterone (18-OH DOC) were measured 24 hours before and again on the day of ACTH infusion. All subjects were studied on the fourth day of a diet containing 135 mEq of sodium and 90 mEq of potassium. There was no statistically significant difference between 8 control subjects and 10 patients with EH in the 7 plasma steroid levels and the SR of 18-OH DOC before ACTH infusion. The mean plasma P response to ACTH was slightly lower in controls than in patients with EH, while that of 17-OH P (in male subjects) was slightly higher. The mean plasma B response was significantly lower after 4 hours of ACTH infusion (p less than 0.01), while that of DOC was significantly higher after 8 hours of ACTH infusion (p less than 0.05) in patients with EH. The mean plasma S rose significantly more in patients with EH (p less than 0.025) at 4 and 8 hours after ACTH infusion. The mean plasma F response to ACTH infusion was slightly lower in patients with EH than in controls. The mean response of 18-OH DOC SR to ACTH infusion was slightly higher in patients with EH than in controls. The mean plasma A response was significantly higher in patients with EH than in controls 4 (p less than 0.05) and 8 hour (p less than 0.001) after an ACTH infusion. These results could be explained in part by abnormalities in the 17- and 11-hydroxylase systems, and that the abnormality in 11-hydroxylation was more pronounced than that in the 17-position. Furthermore, we suspect that the sensitivity of adrenal aldosterone to ACTH might be increased or another accelerated pathway to aldosterone biosynthesis might exist in patients with EH.     

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.     

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.     

Norbormide enhances late steps of steroid-hormone synthesis in rat and mouse adrenal cortex

Norbormide (N) is a vasoconstrictor agent, which acts selectively on the peripheral arteries of the rat, through the activation of the phospholipase C (PLC) cascade and the stimulation of Ca(2+) entrance in the vascular myocytes. Several endogenous vasoconstrictor agent (e.g. angiotensin-II (ANG-II) and endothelin-1 (ET-1)), that stimulate PLC pathway, are also able to enhance aldosterone secretion by the adrenal gland. Hence, we examined the effects of norbormide ((0.5, 1.0 or 5) x 10(-5)M) on corticosteroid-hormone secretion from adrenal slices of rats and mice. Quantitative HPLC assay showed that under basal conditions rat and mouse adrenal quarters secreted progesterone (PROG), 11-deoxycorticosterone (DOC), 18-hydroxy-DOC (18OH-DOC), corticosterone (CORT), 18-hydroxy-corticosterone (18OH-CORT) and aldosterone (ALDO), as well as large amounts of pregnenolone (PREG) when its metabolism was blocked by 10(-5)M cyanoketone. Norbormide concentration-dependently raised the secretion of all post-DOC steroids assayed, decreased progesterone and DOC production, and did not affect pregnenolone release. In conclusion, norbormide is able to enhance late steps of steroid synthesis, i.e. those leading to the transformation of DOC to corticosterone and aldosterone, without affecting early steps. This is an interesting finding because the other main endogenous adrenal secretagogues are known to stimulate both early and late steps of steroid synthesis. The mechanism underlying the selective activating action of norbormide on 11beta- and 18-hydroxylation remains to be investigated.     

Functional expression of the cDNAs encoding rat 11 beta-hydroxylase [cytochrome P450(11 beta)] and aldosterone synthase [cytochrome P450(11 beta, aldo)]

Expression plasmids containing two cDNAs of a rat cytochrome P450(11 beta) family, pcP450(11 beta)-62 [Nonaka, Y., Matsukawa, N., Morohashi, K., Omura, T., Ogihara, T., Teraoka, H. & Okamoto, M. (1989) FEBS Lett. 255, 21-26] and pcP450(11 beta, aldo)-46 [Matsukawa, N., Nonaka, Y., Ying, Z., Higaki, J., Ogihara, T. & Okamoto, M. (1990) Biochem. Biophys. Res. Commun. 169, 245-252], were constructed and introduced into COS-7 cells by electroporation. Enzymatic activities of the expressed cytochromes P450(11 beta) and P450(11 beta, aldo) were determined by using 11-deoxycorticosterone, corticosterone, 18-hydroxy-11-deoxycorticosterone, 18-hydroxycorticosterone, or 19-hydroxy-11-deoxycorticosterone as a substrate. Cytochrome P450(11 beta) catalyzed 11 beta-, 18- and 19-hydroxylations of 11-deoxycorticosterone and 19-oxidation or 19-hydroxy-11-deoxycorticosterone at substantial rates, 18-hydroxylation of corticosterone at a very low rate, but no aldosterone production. Cytochrome P450(11 beta, aldo) catalyzed 11 beta- and 18-hydroxylations of 11-deoxycorticosterone, 18-hydroxylation of corticosterone and aldosterone production from 11-deoxycorticosterone or corticosterone. But neither 19-hydroxylation of 11-deoxycorticosterone nor 19-oxidation of 19-hydroxy-11-deoxycorticosterone was catalyzed by cytochrome P450(11 beta, aldo).     

Relation of the pituitary gland to gonadal hormone effects on the adrenocortical 11beta-hydroxylase system in rats.

Studies were conducted to further examine the mechanisms responsible for gonadal hormone effects on the rat adrenocortical 11beta-hydroxylase system. Despite higher concentrations of cytochrome P-450 and larger 11-deoxycorticosterone (DOC)-induced difference spectra in adrenal mitochondria from females than males, no sex difference in 11beta-hydroxylase activity was observed. The pregnenolone-induced difference spectrum, indicative of cholesterol binding to cytochrome P-450, also was similar in males and females. Testosterone administration to castrated males lowered both 11beta-hydroxylase activity and mitochondrial cytochrome P-450 content. Estradiol produced the opposite effects in castrated females. However, when given to ACTH-replaced hypophysectomized rats, neither testosterone nor estradiol affected cytochrome P-450 levels or the rate of 11beta-hydroxylation. These observations, taken with the known effects of estradiol and testosterone on ACTH secretion in rats and the effects of ACTH on 11beta-hydroxylation, indicate that gonadal hormone effects on the 11beta-hydroxylase system are mediated by ACTH.     

The measurement of 18-hydroxy-11-deoxycorticosterone in human plasma by radioimmunoassay

A radioimmunoassay method for the measurement of plasma levels of 18-hydroxy-11-deoxycorticosterone (18-OH-DOC) has been developed. The antiserum against 18-OH-DOC was produced in rabbits immunized against 18-OH-DOC-3-oxime-bovine serum albumin. Plasma (1–2 ml) was extracted with dichloromethane and chromatographed on paper. The purified extracts were incubated with antiserum at a $\text{1}\text{22,000}$ dilution for $\text{1}\text{2}$ hour at 37°C and for 2 hours at 4°C. Saturated ammonium sulfate was used to separate free from bound 18-OH-DOC. 1, 2-³H-18-OH-DOC was added to all samples to correct for losses and to determine the percent free. Pyridine (0.1%) was added to solvents to maintain the stability of 18-OH-DOC. Recovery after extraction was 58 ± 8 (S.D.)%. The accuracy and precision of the method were acceptable, and a sensitivity of 2 pg per sample enabled the measurement of very low levels of 18-OH-DOC. High specificity was demonstrated by a low blank value (0 ± 0.2 pg) and by demonstrating that alternative paper chromatography separation systems gave results not differing significantly from those obtained by the present method. The mean 8AM plasma 18-OH-DOC level was 8.5 ± 1.2 ng per 100 ml in 18 normotensive control subjects. There was a marked response of plasma 18-OH-DOC to ACTH stimulation and dexamethasone suppression and a significant increase after 3 hours upright posture.     

Metabolism of 11-deoxycorticosterone by hamster adrenal mitochondria.

Metabolism of 11-deoxycorticosterone (DOC) by hamster adrenal mitochondria gives 19-hydroxy-DOC and corticosterone (via 11-hydroxylation) in approximately equal yields. The ratio of 19- to 11-hydroxylation was invariant with changes in concentration of substrate or a competitive inhibitor. It is most likely, therefore, that a single 11,19-hydroxylase catalyzes both oxidations. Both primary products are further oxidized to the corresponding carbonyl analogs, 19-oxo-DOC and 11-dehydrocorticosterone, at rates that are approx. 20% of their rates of formation. The oxidation of 11-dehydrocorticosterone is catalyzed by a dehydrogenase utilizing either NAD or NADP while the oxidation of 19-hydroxy-DOC is catalyzed by an oxidase requiring NADPH. The 11-dehydrocorticosterone is stable in this enzyme preparation while 19-oxo-DOC is metabolized to two additional products, which are tentatively identified as 19-oic-DOC and 19-norcorticosterone. 19-nor-DOC was found to be hydroxylated at a rate that is 20% faster than the rate for DOC under the same conditions. It is therefore possible that 19-norcorticosterone can arise from 19-oic-DOC via decarboxylation to 19-nor-DOC and subsequent 11-hydroxylation, but the kinetics of its formation suggest that it may actually be formed directly from 19-oxo-DOC without free intermediates. 4-Androstene-3,17-dione and 17-hydroxy-DOC were also substrates for this 11,19-hydroxylase, but 18-hydroxy-DOC was not. Maintenance of hamsters on a low sodium diet had no effect on the metabolism of DOC by the isolated adrenal mitochondria.     

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.     

Extraction of corticosterone from cell homogenates and subcellular fractions of the rat adrenal cortex. III. ACTH-induced temporal subcellular redistributions of steroid precursors to corticosterone

Cholesterol, pregnenolone, progesterone, 11-deoxycorticosterone (11-DOC) and corticosterone were quantitated in subcellular fractions isolated from in vivo adrenocorticotropin (ACTH)-stimulated rat adrenal zona fasciculata/reticularis. Six adrenal subcellular fractions separated by discontinuous sucrose gradient centrifugation (lipid, 0.125 M sucrose, cytosolic, microsomal, mitochondrial and nuclear) were extracted with alkaline ether/ethanol and assayed by high pressure liquid chromatography (HPLC). Lipid fractions contained the major cholesterol stores, while most pregnenolone and progesterone was found in lipid, microsomal and mitochondrial fractions. The 0.125 M sucrose and cytosol fractions together contained approximately 75% of the total 11-DOC and corticosterone. The five steroids were only present in small amounts in organelle fractions containing steroidogenic enzymes. Homogenate and lipid fraction cholesterol decreased between 10 and 15 min and again 30 min after ACTH injection. In the homogenate, lipid, microsomal and mitochondrial fractions, pregnenolone and progesterone were increased after ACTH injection; peak pregnenolone and progesterone concentrations were often measured in adrenal gland sucrose, cytosolic, microsomal and mitochondrial fractions 15 to 20 min after rats were injected with ACTH. Although ACTH increased 11-DOC and corticosterone in all but the mitochondrial and nuclear fractions, the sucrose, cytosolic and microsomal 11-DOC, and cytosolic corticosterone increased most dramatically. In many fractions, peak 11-DOC and corticosterone concentrations were most often observed between the 10 and 15 min periods and again at 30 min.     

Serum concentrations of deoxycorticosterone in women during the luteal phase of the ovarian cycle are not suppressed by dexamethasone treatment

We determined the serum levels of deoxycorticosterone (DOC) in plasma of six healthy, apparently ovulatory women during the mid-follicular and mid-luteal phases of their ovarian cycles; and we evaluated the effect of dexamethasone (1 mg by mouth) on the concentrations of DOC and cortisol in serum at times when plasma progesterone levels were high or low. The serum levels of DOC, unlike those of cortisol, did not vary significantly in single blood samples obtained in the morning (8-10 a.m.) and afternoon (3-5 p.m.); and serum DOC levels in women were significantly higher (P less than 0.05) during the mid-luteal phase than during the mid-follicular phase of the cycle. There were unmistakable diurnal variations in serum levels of cortisol, and cortisol concentrations were reduced to less than 20% of pretreatment levels after the ingestion of 1 mg dexamethasone during the mid-follicular or mid-luteal phase. The serum concentrations of DOC were reduced only to approx 70% of pretreatment levels after dexamethasone ingestion during the follicular phase. The serum levels of DOC did not decline significantly after administration of dexamethasone during the mid-luteal phase, when progesterone levels in serum are high (14-16 ng/ml). Blood samples also were obtained at hourly intervals during the 24 h before and after dexamethasone administration in one woman during the follicular phase and in another woman the during the early luteal phase (progesterone levels = 1-3 ng/ml) of the ovarian cycle. DOC levels (pre-dexamethasone) fluctuated in synchrony with those of cortisol in the woman studied during the follicular phase but not in the woman studied during the early luteal phase of the cycle. In the post-dexamethasone period, plasma cortisol levels were suppressed for at least 24 h in both women whereas DOC levels were decreased only partially. We conclude that plasma DOC is derived from both adrenal secretion and from extraadrenal 21-hydroxylation of progesterone--the latter source of DOC is not affected by dexamethasone suppression of ACTH secretion.     

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.