Conversion Of Deoxycorticosterone Research Articles

Neurosteroids as Endogenous Regulators of Seizure Suscepibility.

Stress-induced Deoxycorticosterone-derived Neurosteroids Modulate GABAA Receptor Function and Seizure Susceptibility Reddy DS, Rogawski MA J Neurosci 2002;22:3795–3805 Stress affects seizure susceptibility in animals and humans, but the underlying mechanisms are obscure. Here we provide evidence that γ-aminobutyric acid subunit A (GABAA) receptor-modulating neurosteroids derived from deoxycorticosterone (DOC) play a role in stress-related changes in seizure control. DOC, an adrenal steroid, the synthesis of which is enhanced during stress, undergoes sequential metabolic reduction by 5α-reductase and 3α-hydroxysteroid oxidoreductase to form 5α-dihydrode-oxycorticosterone (DHDOC) and allotetrahydrodeoxycorti-costerone (THDOC), a GABAA receptor-modulating neurosteroid with anticonvulsant properties. Acute swim stress in rats significantly elevated plasma THDOC concentrations and raised the pentylenetetrazol (PTZ) seizure threshold. Small systemic doses of DOC produced comparable increases in THDOC and PTZ seizure thresholds. Pretreatment with finasteride, a 5α-reductase inhibitor that blocks the conversion of DOC to DHDOC, reversed the antiseizure effects of stress. DOC also elevated plasma THDOC levels and protected mice against PTZ, methyl-6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate, picrotoxin, and amygdala-kindled seizures in mice (ED50 values, 84–97 mg/kg). Finasteride reversed the antiseizure activity of DOC (ED50, 7.2 mg/kg); partial antagonism also was obtained with indomethacin (100 mg/kg), an inhibitor of 3α-hydroxysteroid oxidoreductase. Finasteride had no effect on seizure protection by DHDOC and THDOC, whereas indomethacin partially reversed DHDOC but not THDOC. DHDOC, like THDOC, potentiated GABA-activated Cl– currents in cultured hippocampal neurons (≤1 μ) and directly activated GABAA receptor currents (≤1 μ M), compatible with a role for DHDOC in the antiseizure activity of DOC. DOC is a mediator of the physiological effects of acute stress that could contribute to stress-induced changes in seizure susceptibility through its conversion to neurosteroids with modulatory actions on GABAA receptors including THDOC and possibly also DHDOC. Enhanced Neurosteroid Potentiation of Ternary GABAA Receptors Containing the δ Subunit Wohlfarth KM, Bianchi MT, Macdonald RL J Neurosci 2002;22:1541–1549 Attenuated behavioral sensitivity to neurosteroids has been reported for mice deficient in the γ-aminobutyric acid subunit A [GABA(A)]-receptor δ subunit. We therefore investigated potential subunit-specific neurosteroid pharmacology of the following GABAA-receptor isoforms in a transient expression system: α1β372L, α1β38, α6β372L, and α6β38. Potentiation of submaximal GABAA-receptor currents by the neurosteroid tetrahydrodeoxycorticosterone (THDOC) was greatest for the α1β38 isoform. Whole-cell GABA concentration-response curves performed with and without low concentrations (30 n M) of THDOC revealed enhanced peak GABAA-receptor currents for isoforms tested without affecting the GABA EC50. The α1β38 currents were enhanced the most (>150%), whereas the other isoform currents were enhanced 15–50%. At a higher concentration (1 μ M), THDOC decreased peak α1β382L-receptor current amplitude evoked by GABA (1 m M) concentration jumps and prolonged deactivation but had little effect on the rate or extent of apparent desensitization. Thus the polarity of THDOC modulation depended on GABA concentration for α1β382L GABAA receptors. However, the same protocol applied to α1β38 receptors resulted in peak current enhancement by THDOC of >800% and prolonged deactivation. Interestingly, THDOC induced pronounced desensitization in the minimally desensitizing α1β38 receptors. Single-channel recordings obtained from α1β38 receptors indicated that THDOC increased the channel-opening duration, including the introduction of an additional longer duration open state. Our results suggest that the GABAA-receptor delta subunit confers increased sensitivity to neurosteroid modulation and that the intrinsic gating and desensitization kinetics of α1β38 GABAA receptors are altered by THDOC.

Stress-Induced Deoxycorticosterone-Derived Neurosteroids Modulate GABAAReceptor Function and Seizure Susceptibility

Stress affects seizure susceptibility in animals and humans, but the underlying mechanisms are obscure. Here, we provide evidence that GABA_A receptor-modulating neurosteroids derived from deoxycorticosterone (DOC) play a role in stress-related changes in seizure control. DOC, an adrenal steroid whose synthesis is enhanced during stress, undergoes sequential metabolic reduction by 5α-reductase and 3α-hydroxysteroid oxidoreductase to form 5α-dihydrodeoxycorticosterone (DHDOC) and allotetrahydrodeoxycorticosterone (THDOC), a GABA_Areceptor-modulating neurosteroid with anticonvulsant properties. Acute swim stress in rats significantly elevated plasma THDOC concentrations and raised the pentylenetetrazol (PTZ) seizure threshold. Small systemic doses of DOC produced comparable increases in THDOC and PTZ seizure threshold. Pretreatment with finasteride, a 5α-reductase inhibitor that blocks the conversion of DOC to DHDOC, reversed the antiseizure effects of stress. DOC also elevated plasma THDOC levels and protected mice against PTZ, methyl-6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate, picrotoxin, and amygdala-kindled seizures in mice (ED₅₀ values, 84–97 mg/kg). Finasteride reversed the antiseizure activity of DOC (ED₅₀, 7.2 mg/kg); partial antagonism was also obtained with indomethacin (100 mg/kg), an inhibitor of 3α-hydroxysteroid oxidoreductase. Finasteride had no effect on seizure protection by DHDOC and THDOC, whereas indomethacin partially reversed DHDOC but not THDOC. DHDOC, like THDOC, potentiated GABA-activated Cl⁻ currents in cultured hippocampal neurons (≤1 μm) and directly activated GABA_Areceptor currents (≥1 μm), compatible with a role for DHDOC in the antiseizure activity of DOC. DOC is a mediator of the physiological effects of acute stress that could contribute to stress-induced changes in seizure susceptibility through its conversion to neurosteroids with modulatory actions on GABA_A receptors including THDOC and possibly also DHDOC.

Stress-induced deoxycorticosterone-derived neurosteroids modulate GABA(A) receptor function and seizure susceptibility.

Stress affects seizure susceptibility in animals and humans, but the underlying mechanisms are obscure. Here, we provide evidence that GABA(A) receptor-modulating neurosteroids derived from deoxycorticosterone (DOC) play a role in stress-related changes in seizure control. DOC, an adrenal steroid whose synthesis is enhanced during stress, undergoes sequential metabolic reduction by 5alpha-reductase and 3alpha-hydroxysteroid oxidoreductase to form 5alpha-dihydrodeoxycorticosterone (DHDOC) and allotetrahydrodeoxycorticosterone (THDOC), a GABA(A) receptor-modulating neurosteroid with anticonvulsant properties. Acute swim stress in rats significantly elevated plasma THDOC concentrations and raised the pentylenetetrazol (PTZ) seizure threshold. Small systemic doses of DOC produced comparable increases in THDOC and PTZ seizure threshold. Pretreatment with finasteride, a 5alpha-reductase inhibitor that blocks the conversion of DOC to DHDOC, reversed the antiseizure effects of stress. DOC also elevated plasma THDOC levels and protected mice against PTZ, methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate, picrotoxin, and amygdala-kindled seizures in mice (ED50 values, 84-97 mg/kg). Finasteride reversed the antiseizure activity of DOC (ED50, 7.2 mg/kg); partial antagonism was also obtained with indomethacin (100 mg/kg), an inhibitor of 3alpha-hydroxysteroid oxidoreductase. Finasteride had no effect on seizure protection by DHDOC and THDOC, whereas indomethacin partially reversed DHDOC but not THDOC. DHDOC, like THDOC, potentiated GABA-activated Cl- currents in cultured hippocampal neurons (< or =1 microm) and directly activated GABA(A) receptor currents (> or =1 microm), compatible with a role for DHDOC in the antiseizure activity of DOC. DOC is a mediator of the physiological effects of acute stress that could contribute to stress-induced changes in seizure susceptibility through its conversion to neurosteroids with modulatory actions on GABA(A) receptors including THDOC and possibly also DHDOC.

Regulation of aldosterone biosynthesis: the end of the road?

1. Because aldosterone is the minority hormone of the adrenal cortex, its proper function depends on protective physiological mechanisms. These include a particular site of aldosterone biosynthesis in the zona glomerulosa of the adrenal cortex as well as a complex multifactorial control system, which adapts aldosterone production to acute and chronic changes in body sodium and potassium content, irrespective of pituitary adrenocorticotropic hormone (ACTH) secretion. 2. Over the past decade, an important element of these mechanisms has been identified in the form of the enzyme involved in the final steps of aldosterone biosynthesis. In species such as the human, rat and mouse, the conversion of deoxycorticosterone to aldosterone is catalysed by an isozyme (CYP11B2) of cytochrome P450(11) beta (CYP11B1). The gene encoding this enzyme is expressed only in the zona glomerulosa. Its transcription is enhanced by sodium deficiency and potassium intake, but is suppressed by long-term administration of high doses of ACTH. 3. In contrast, the gene encoding CYP11B1 (i.e. the major (non-aldosterone-producing) type of the enzyme) is expressed mainly in the zona fasciculata and its expression depends on physiological concentrations of ACTH. 4. In other animal species (cattle, pig, sheep), the major forms of cytochrome P450(11) beta have an inherent aldosterone-synthesizing activity, which is, however, selectively suppressed in mitochondria of zona fasciculata cells. 5. The elucidation of the mechanisms involved in this suppression or of those mediating alterations in CYP11B2 expression in response to physiological stimuli may be important areas of future research on the regulation of aldosterone biosynthesis.

Aldosterone Secretion: A Molecular Perspective

The major mineralocorticoid hormone aldosterone is secreted from the zona glomerulosa of the adrenal cortex. Aldosterone is synthesized from cholesterol via a series of hydroxylations and oxidations. The enzymes involved in these reactions are mostly members of the cytochrome P450 superfamily. The final steps of this pathway, the conversion of 11-deoxycorticosterone (DOC) to aldosterone, require conversion via the intermediates 18-hydroxy-DOC or corticosterone and 18-hydroxycorticosterone. There are significant differences between species in the number of genes that encode the P450 11β-related enzymes ( CYP11B ) involved in these steps and the zonal distribution of their expression. One enzyme is capable of 11-hydroxylation, 18-hydroxylation, and 18-oxidation of DOC to aldosterone. The genetic basis of four diseases—congenital adrenal hyperplasia due to 11β-hydroxylase deficiency, glucocorticoid-remediable aldosteronism, aldosterone synthase deficiency type I and type II—is explicable by mutations in these cytochrome P450 11β-related genes. (Trends Endocrinol Metab 1997;8:346–354). © 1997, Elsevier Science Inc.

Artificial mutations in P450c11AS (aldosterone synthase) can increase enzymatic activity: a model for low-renin hypertension?

The conversion of 11-deoxycorticosterone (DOC) to aldosterone is catalyzed by a single enzyme, termed P450c11AS, which has 11 beta-hydroxylase, 18-hydroxylase and 18-oxidase activities. The normotensive Dahl salt-resistant (R) rat has two mutation in P450c11AS that increase its aldosterone synthase activity. If such a mutation were to occur in human patients the predicted phenotype would be low-renin hypertension with elevated ratios of plasma aldosterone to plasma renin activity. Before searching for P450c11AS mutations in such patients we sought to determine if mutations in human P450c11AS could increase enzymatic activity in a fashion analogous to the Dahl R rat. We used site-directed mutagenesis of the human P450c11AS cDNA to create the mutants Glu 136-->Asp, Lys 251-->Arg and the combination of the two; these mutations correspond to those seen in the Dahl R rat. Cells transfected with these mutant human P450c11AS sequences could convert [14C]DOC to corticosterone, 18OH-corticosterone, and aldosterone. In particular the Lys 251-->Arg mutant produced 4 times as much 18OH-corticosterone and 50-80% more aldosterone than the wild type. These data show that mutations of human P450c11AS can increase enzymatic activity, suggesting that such mutations could, in theory, be the basis of some forms of human low-renin hypertension.

Artificial mutations in P450c11AS (aldosterone synthase) can increase enzymatic activity: a model for low-renin hypertension?

The conversion of 11-deoxycorticosterone (DOC) to aldosterone is catalyzed by a single enzyme, termed P450c11AS, which has 11 beta-hydroxylase, 18-hydroxylase and 18-oxidase activities. The normotensive Dahl salt-resistant (R) rat has two mutation in P450c11AS that increase its aldosterone synthase activity. If such a mutation were to occur in human patients the predicted phenotype would be low-renin hypertension with elevated ratios of plasma aldosterone to plasma renin activity. Before searching for P450c11AS mutations in such patients we sought to determine if mutations in human P450c11AS could increase enzymatic activity in a fashion analogous to the Dahl R rat. We used site-directed mutagenesis of the human P450c11AS cDNA to create the mutants Glu 136-->Asp, Lys 251-->Arg and the combination of the two; these mutations correspond to those seen in the Dahl R rat. Cells transfected with these mutant human P450c11AS sequences could convert [14C]DOC to corticosterone, 18OH-corticosterone, and aldosterone. In particular the Lys 251-->Arg mutant produced 4 times as much 18OH-corticosterone and 50-80% more aldosterone than the wild type. These data show that mutations of human P450c11AS can increase enzymatic activity, suggesting that such mutations could, in theory, be the basis of some forms of human low-renin hypertension.

Separate induction of the two isozymes of cytochrome P-450 11 β in rat adrenal zona glomerulosa cells

In the rat adrenal cortex, two isozymes of cytochrome P-450 11 β (CYP11B1 and CYP11B2) have been identified. They are encoded by two different genes with a homology much higher in their coding than in their 5′-flanking regions. CYP11B1 is found in all the zones of the gland and catalyzes a single hydroxylation of deoxycorticosterone (DOC) in the 11β- or the 18-position. CYP11B2 is produced exclusively in the zona glomerulosa and catalyzes all three reactions involved in the conversion of DOC to aldosterone. In vivo and in vitro, the expression of the genes encoding CYP11B1 and CYP11B2 is regulated by two separate control systems which appear to operate both independently and interdependently. In vivo, zona glomerulosa expression of CYP11B1 was enhanced by ACTH treatment or potassium depletion and was lowered by potassium repletion. CYP11B2 expression disappeared upon potassium depletion or ACTH treatment, but reappeared during potassium repletion. In vitro, only CYP11B1 activity was detectable and responsive to ACTH treatment in zona glomerulosa cells cultured at a potassium concentration of 6.4 mmol/1. Aldosterone biosynthetic activity and mRNA encoding CYP11B2 could be detected only after at least 1 day of exposure to a high extracellular potassium concentration (⩾12 mmol/1).

Rapid regulation of corticotropin-releasing hormone gene transcription in vivo.

Regulation of corticotropin-releasing hormone (CRH) gene expression in vivo was assessed via in situ hybridization histochemistry, using probes directed against an intronic sequence of the CRH gene. Initial characterization of the CRH intron (CRHin) probe revealed specific localization of signal to the nuclear compartment of neurons in the medial parvocellular paraventricular hypothalamus, which are known to produce CRH peptide and mRNA. Abundance of CRHin signal was low, commensurate with a low resting pool of CRH heteronuclear RNA (hnRNA), representing CRH primary transcript. Regulation of CRH hnRNA levels was assessed after acute glucocorticoid synthesis blockade by injection of metyrapone. Metyrapone inhibits the conversion of 11-deoxycorticosterone to corticosterone, thereby rapidly depleting glucocorticoids and serving as a discrete stimulus for hypothalamo-pituitary-adreno-cortical activation. Plasma hormone measurements verified the efficacy of treatment, as metyrapone-treated rats showed extremely low basal corticosterone levels at all postinjection time points, while exhibiting progressive increases in plasma ACTH release over the 60-min postinjection period. CRH hnRNA levels were markedly increased 15-30 min after metyrapone injection, consistent with a rapid induction of CRH gene transcription in response to the stimulatory event. CRH mRNA, on the other hand, did not exceed control levels until 60 min post metyrapone, illustrative of a temporal lag between transcriptional changes and detectable changes in mRNA pools. Additional sections from metyrapone-and vehicle-treated rats were hybridized with probes complementary to mRNA encoding the immediate-early gene c-fos. c-fos was not present under unstimulated conditions yet was rapidly induced upon metyrapone treatment or vehicle injection (15 min).(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Evidence that corticosterone is not an obligatory intermediate in aldosterone biosynthesis in the rat adrenal

CGS 16949A is a potent inhibitor of aromatase in vitro with an IC50 of 0.03 microM for the inhibition of LH-stimulated estrogen biosynthesis in hamster ovaries. In vivo, CGS 16949A leads to sequelae of estrogen deprivation (e.g. regression of DMBA-induced mammary tumors) without causing adrenal hypertrophy in adult rats. To complement these in vitro and in vivo findings, the effect of CGS 16949A on adrenal steroid biosynthesis in rats was investigated in vitro and in vivo. The surprising finding in vitro was that CGS 16949A inhibited aldosterone biosynthesis (IC50 = 1 microM) at concentrations 100 times lower than those for inhibition of corticosterone biosynthesis (IC50 = 100 microM). Moreover, deoxycorticosterone (DOC) concentrations were elevated at all concentrations of CGS 16949A which inhibited aldosterone synthesis. The classical biosynthetic pathway for aldosterone is DOC----corticosterone----18-OH-corticosterone----aldosterone. Thus inhibition of aldosterone biosynthesis, reflected in DOC accumulation, without affecting corticosterone concentrations, indicates that corticosterone is not an obligatory intermediate in the conversion of DOC to aldosterone in the rat. In vivo, CGS 16949A showed a suppression of plasma aldosterone in ACTH-stimulated male rats at doses which did not significantly affect plasma corticosterone. In conclusion, aldosterone measured both in vitro and in vivo must be derived primarily from a biosynthetic pathway in which corticosterone is not obligatory intermediate.

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

Highly purified cytochrome P-450 11β-/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-11β-hydroxylase (IgG) before assay. It is concluded that cytochrome P-450 11β-/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: C 21 H 30 O 3 + 3 NADPH + 3 H + + 3 O 2 → C 21 H 28 O 5 + 3 NADP + + 4 H 2 O

Effects of metyrapone and etomidate on adrenal function and growth rate in female rats.

Two inhibitors of adrenal steroidogenesis were examined to determine whether the growth rate of female rats could be improved by lowering circulating plasma corticosterone concentrations. The first inhibitor, etomidate, is a potent narcotic agent and was found to have no effect on plasma corticosterone and deoxycorticosterone (DOC) concentrations at sub-narcotic doses. Growth rate, food intake, food conversion efficiency and adrenal weight were also unaffected by the drug. The second inhibitor, metyrapone, was shown in acute studies to have two distinct actions. In 6-week-old female rats moderate doses of metyrapone (50 mg/kg) had a stimulatory action resulting in increased plasma DOC and corticosterone concentrations. Higher doses of metyrapone (150-300 mg/kg) were increasingly less selective, causing an increase in plasma concentration of DOC, but attenuating the increase in corticosterone concentration, presumably by inhibiting the 11 beta-hydroxylase enzyme which allows the conversion of DOC to corticosterone. In adult rats (greater than 12 weeks old) the classical response to metyrapone was observed. Plasma DOC concentrations were increased, while corticosterone levels were reduced in a dose-dependent manner. In 5-week-old rats treated chronically, metyrapone (300 mg/kg) had no effect on plasma corticosterone, but increased plasma DOC concentration, depressed food intake and reduced growth rate. In 8-week-old rats treated chronically, the high dose of metyrapone (300 mg/kg) required to lower peak plasma corticosterone concentrations produced toxic effects resulting in the death of three animals. The remaining animals recovered rapidly, and gained more weight than controls over the final 6 days of the experiment. However, this experiment was terminated before the treated animals were able to catch up with the controls.(ABSTRACT TRUNCATED AT 250 WORDS)

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 20α-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 20α and/or 3α/β and 5α 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 11βand 18-hydroxylation yielding corticosterone and 18-hydroxyDOC. A minor reductive metabolism was found. Both cultures produced 6β-hydroxyDOC. These results demonstrate that ACTH is needed to maintain the efficiency of the 11β/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.

Metabolism of deoxycorticosterone and deoxycorticosterone sulfate in men and women.

In this investigation we found that little of intravenously infused [14C]deoxycorticosterone (DOC) was converted to [14C]DOC-SO4 that entered plasma. Moreover little of intravenously infused [3H]DOC-SO4 was metabolized by way of DOC except by intestinal bacterial enzymes. However evidence was obtained that plasma DOC is converted to DOC-SO4 in liver, but little of the DOC-SO4 formed in liver escapes into blood; rather the DOC-SO4 enters bile and in the intestine is converted, in part, to progesterone (or metabolites thereof) by the action of bacterial enzymes. The estimated intrahepatic fractional conversion of DOC to DOC-SO4 was significantly greater in premenopausal women (0.72 +/- 0.118, mean +/- SEM) than in men (0.28 +/- 0.036, P less than 0.005).

Mitochondrial 11 β hydroxylation and adrenal morphology in rats bearing a mammotropic, ACTH-secreting pituitary tumor

The function and structure of adrenal mitochondria has been studied in rats bearing an ACTH, growth hormone and prolactin secreting tumor (MtT-F4). Basal levels of 11 β hydroxylation of deoxycorticosterone (DOC) in incubated mitochondria were higher in MtT-F4 than in controls ( P < 0.001). The addition of NADH and calcium to controls increased the conversion of DOC to corticosterone ( P < 0.001) but no change was observed in MtT-F4. With addition of NADPH plus calcium, the increase in controls was of a much higher magnitude than that produced in MtT-F4 ( P < 0.05). Calcium alone diminished the high levels of hydroxylation in MtT-F4. Measurement of mitochondrial swelling showed that in the presence of albumin and at 37°C swelling rates in MtT-F4 and controls were similar; however, large amplitude swelling developed in MtT-F4 when albumin was omitted and temperature was lowered to 25°C. ATPase activity was not different in mitochondria from both groups of rats either in the presence or absence of magnesium. The depressive effect of calcium, an uncoupler of oxidative phosphorylation, showed that some form of energy was needed for the high rate of basal hydroxylation in MtT-F4. The diminished response to the nucleotides indicated that probably both the P 450 hydroxylation chain and the transhydrogenase enzyme are impaired in MtT-F4. Electron microscopy of whole adrenal cortex showed a general appearance of hyperfunction. Mitochondria of zona glomerulosa contained inclusions of high electron density, and the outer membrane showed a multillaminar appearance. In zona fasciculata mitochondria showed normal structure, but different electronic density and individual variation in the number of cristae. In contrast, mitochondria of zona reticularis showed normal morphology.

A comparison of steroidogenesis in vitro in two teleosts, the marine herring, Clupea, and the freshwater Atlantic salmon, Salmo.

SUMMARY Cortisol was the principal steroid biotransformed from [16-3H]pregnenolone incubated with interrenal tissues of the Atlantic herring, Clupea harengus harengus, and of the Atlantic salmon, Salmo salar. Corticosterone was the preferred product from [4-14C]progesterone in herring, and cortisol in salmon. In these two teleosts 3H:14C recrystallization ratios of cortisol produced from the equimolar amounts of precursors at various times of incubation suggested that pregnenolone was the better precursor of cortisol and that the favoured pathway to cortisol was via 17-hydroxylated rather than 17-deoxy intermediates. It was demonstrated that interrenal tissues of herring and salmon both have the capacity to 17-hydroxylate corticosterone to cortisol; aldosterone was not detected in any of the interrenal incubates. The 17-hydroxylation of 11-deoxycorticosterone to 11-deoxycortisol was shown in vitro by interrenal tissues of the Atlantic salmon, a conversion that has not been previously demonstrated in fish or in normal tissues of other vertebrates. When interrenal tissue of Atlantic salmon was incubated with equimolar amounts of [1,2-3H] 11-deoxycorticosterone plus [4-14C]corticosterone, there was more conversion of 11-deoxycorticosterone to 11-deoxycortisol (0·39%) than of corticosterone to cortisol (0·12%) and only a small amount (1·0%) of the 3H-labelled precursor was associated with corticosterone after 4 h incubation. Biotransformed cortisol was isolated from both herring and salmon interrenals incubated with each of the following pairs of equimolar substrates: [3H]21-deoxycortisol plus [4-14C]11-deoxycortisol, [3H]21-deoxycortisol plus [4-14C]corticosterone, and [7-3H]11-deoxycortisol plus [4-14C]corticosterone. Isotope ratios of product cortisol suggested that, under the in-vitro conditions employed, substrate efficiency to cortisol synthesis was in the order: 11-deoxycortisol ⪢ 21-deoxycortisol &gt; corticosterone. The results indicated that steroid hydroxylations at C-11, C-17 and C-21 could occur in any order in vitro in interrenals of herring and of salmon.

ALDOSTERONE STIMULATION IN VITRO

ABSTRACT Tritiated precursors of corticosteroids were incubated with rat adrenal sections. At the end of the incubation, tritium-labelled aldosterone and corticosterone were determined by double isotope dilution procedures. When corticosterone-[3H], deoxycorticosterone-[3H], progesterone-[3H] or pregnenolone-[3H] were used as substrates, 2–5% of the total radioactivity in the incubation medium could be recovered in the aldosterone fraction, while 20–25% of the latter three substrates were incorporated into corticosterone. Potassium ions, ammonium ions, angiotensin II and ACTH markedly stimulated the biosynthesis of unlabelled aldosterone from endogenous precursors. However, they did not influence the amounts of radioactive substrate converted to aldosterone or corticosterone. NADP and glucose 6-phosphate stimulated both the total production of aldosterone and corticosterone and the conversion of deoxycorticosterone-[3H] and of pregnenolone-[3H] to corticosterone. Only minute amounts of tritiated cholesterol were converted to corticosteroids. Angiotensin II and ammonium ions significantly stimulated the conversion of cholesterol-[3H] to aldosterone, while ACTH, potassium ions and particularly NADP and glucose 6-phosphate enhanced its incorporation into both aldosterone and corticosterone. These findings indicate that, like ACTH and NADPH2, monovalent cations and angiotensin act mainly on the conversion of cholesterol to pregnenolone and do not directly stimulate hydroxylation or dehydrogenation in position 18.