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.     

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.     

Aldosterone biosynthesis and cytochrome P-45011 beta: evidence for two different forms of the enzyme in rats

Whereas cytochrome P-45011 beta has been recently shown to catalyze the two-step conversion of corticosterone to aldosterone in the bovine and porcine adrenal cortex, the identity of the enzyme involved in the two final steps of aldosterone biosynthesis in the rat adrenal cortex is as yet unknown. Mitochondria from capsular adrenals of sodium-deficient, potassium-replete rats converted corticosterone to 18-hydroxycorticosterone and aldosterone at markedly higher rates than mitochondria from capsular adrenals of sodium-replete, potassium-deficient rats. However, the same preparations exhibited no difference in the 11 beta-hydroxylase activity, i.e. the conversion of deoxycorticosterone to corticosterone. Only mitochondria of zona glomerulosa from rats with stimulated aldosterone biosynthesis contained a 49K protein which showed a strong cross-reactivity with a monoclonal antibody raised against purified bovine cytochrome P-45011 beta. By contrast, a crossreactive protein with a molecular weight of 51K was found in mitochondria of zona fasciculata and in mitochondria of zona glomerulosa from rats with a suppressed aldosterone biosynthesis. These findings indicate the existence of two different forms of cytochrome P-45011 beta in the rat adrenal cortex, with only one of them, i.e. the 49K form, being capable of catalyzing the two final steps of aldosterone biosynthesis in situ.     

Isolation of aldosterone synthase cytochrome P-450 from zona glomerulosa mitochondria of rat adrenal cortex

A cytochrome P-450 capable of producing aldosterone from 11-deoxycorticosterone was purified from the zona glomerulosa of rat adrenal cortex. The enzyme was present in the mitochondria of the zona glomerulosa obtained from sodium-depleted and potassium-repleted rats but scarcely detected in those from untreated rats. It was undetectable in the mitochondria of other zones of the adrenal cortex from both the treated and untreated rats. The cytochrome P-450 was distinguishable from cytochrome P-45011 beta purified from the zonae fasciculata-reticularis mitochondria of the same rats. Molecular weights of the former and the latter cytochromes P-450, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, were 49,500 and 51,500, respectively, and their amino acid sequences up to the 20th residue from the N terminus were different from each other at least in one position. The former catalyzed the multihydroxylation reactions of 11-deoxycorticosterone giving corticosterone, 18-hydroxydeoxycorticosterone, 18-hydroxycorticosterone, and a significant amount of aldosterone as products. On the other hand, the latter catalyzed only 11 beta- and 18-hydroxylation reactions of the same substrate to yield either corticosterone or 18-hydroxydeoxycorticosterone. Thus, at least two forms of cytochrome P-450, which catalyze the 11 beta- and 18-hydroxylations of deoxycorticosterone, exist in rat adrenal cortex, but aldosterone synthesis is catalyzed only by the one present in the 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.     

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).     

Cloning and expression of a cDNA for human cytochrome P-450aldo as related to primary aldosteronism

A cDNA clone encoding human aldosterone synthase cytochrome P-450 (P-450aldo) has been isolated from a cDNA library derived from human adrenal tumor of a patient suffering from primary aldosteronism. The insert of the clone contains an open reading frame encoding a protein of 503 amino acid residues together with a 3 bp 5'-untranslated region and a 1424 bp 3'-untranslated region to which a poly(A) tract is attached. The nucleotide sequence of P-450aldo cDNA is 93% identical to that of P-450(11) beta cDNA. Catalytic functions of these two P-450s expressed in COS-7 cells are very similar in that both enzymes catalyze the formation of corticosterone and 18-hydroxy-11-deoxycorticosterone using 11-deoxycorticosterone as a substrate. However, they are distinctly different from each other in that P-450aldo preferentially catalyzes the conversion of 11-deoxycorticosterone to aldosterone via corticosterone and 18-hydroxycorticosterone while P-450(11)beta substantially fails to catalyze the reaction to form aldosterone. These results suggest that P-450aldo is a variant of P-450(11)beta, but this enzyme is a different gene product possibly playing a major role in the synthesis of aldosterone at least in a patient suffering from primary aldosteronism.     

Differences between adrenal adenoma causing primary aldosteronism and other adrenal tissues in the incorporation of labeled steroid precursors into their products

The incorporation and conversion of several labeled steroid precursors into their products were examined in slices of adrenal tissue from two patients with primary aldosteronism and compared with that in “normal” adrenal tissue and adrenal tissues from a patient with Gushing's syndrome. The products of the incorporation were separated by Sephadex LH-20 column chromatography. The major products of conversion in the adenomatous tissue of primary aldosteronism were 18-hydroxycorticosterone and lesser amounts of aldosterone. Smaller amounts of 18-hydroxycorticosterone were isolated from all other adrenal tissues studied. No aldosterone could be recovered after incubating any of the adrenal tissue studied with labeled 18-hydroxy-11-deoxycorticosterone or 18-hydroxycorticosterone as precursor steroid. These $\text{in vitro}$ results seem to suggest that there is increased 18-hydroxylation in the adenoma of primary aldosteronism compared with other tissues and that relatively more 18-hydroxycorticosterone is produced in such tissue than aldosterone.     

Chromatographic system for the simultaneous measurement of plasma 18-hydroxy-11-deoxycorticosterone and 18-hydroxycorticosterone by radioimmunoassay: reference data for neonates and infants and its application in aldosterone-synthase deficiency

A new chromatographic system for the steroid precursor separation and a sensitive radioimmunoassay system for the subsequent measurement of 18-hydroxy-11-deoxycorticosterone and 18-hydroxycorticosterone has been developed. 18-Hydroxy-11-deoxycorticosterone and 18-hydroxycorticosterone were extracted with methylene chloride and separated from cross-reacting steroids by Sephadex LH-20 column chromatography. Anti-18-hydroxy-11-deoxycorticosterone and anti-18-hydroxycorticosterone antibodies raised in rabbits were used. The lower detection limit of the assay is 0.03 nmol/l and 0.128 nmol/l for 18-hydroxy-11-deoxycorticosterone and 18-hydroxycorticosterone, respectively. Normal values for this assay in 128 healthy neonates and infants aged 0-5 months were established as a basis for the early hormonal diagnosis of aldosterone synthase deficiency types I and II. Its application for the diagnosis of aldosterone synthase deficiency is demonstrated in two patients with homozygous mutation/deletion in the encoding CYP11B2 gene.     

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.     

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.     

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.     

Molecular cloning and expression of cDNAS encoding rat aldosterone synthase: variants of cytochrome P-450(11 beta)

Two distinct forms of cDNA encoding rat aldosterone synthase were cloned from an adrenal capsular tissue cDNA library. The deduced amino acid sequences showed that one of the enzymes (P-450(11 beta),aldo-1) had a long extension peptide composed of 34 amino acid residues while the other (P-450(11 beta),aldo-2) had an extension peptide identical to that of rat P-450(11 beta). Glu at the 320th position of P-450(11 beta),aldo-1 was replaced with Lys in P-450(11 beta),aldo-2. The amino acid sequence of the aldosterone synthase was highly homologous (81%) to rat P-450(11 beta). Constructed expression vector containing the cDNA for extension peptide of P-450(11 beta) and the mature protein of P-450(11 beta),aldo-1 was transfected into COS-7 cells. The cells converted 11-deoxycorticosterone into corticosterone, 18-hydroxycorticosterone, and aldosterone.     

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.     

18-Hydroxy-11-deoxycortisol: a new steroid isolated from incubations of the adrenal with 11-deoxycortisol

Cortisol has been shown to be metabolized in the zona glomerulosa of the adrenal gland through the same pathway involving the cytochrome P-450, corticosterone methyl oxidase by which corticosterone is transformed to 18-hydroxycorticosterone and aldosterone. When cortisol is the precursor, 18-hydroxycortisol and 18-oxocortisol are formed. 18-Hydroxycortisol can also be made at a similar rate in the bovine zona fasciculata and reticularis as in the zona glomerulosa. We studied the possibility that the formation of 18-hydroxycortisol in the zona fasciculata and reticularis might be through a different pathway involving initial 18-hydroxylation of 11-deoxycortisol before 11 beta-hydroxylation. Rat adrenal capsules or cores were incubated with 10 micrograms of cortisol or 11-deoxycortisol and the formation of 18-hydroxycortisol was measured by radioimmunoassay. Both capsules and cores transformed 11-deoxycortisol to 18-hydroxycortisol, but cortisol was only transformed in the capsular portion. Sixty-two rat adrenals were incubated with 10 mg of 11-deoxycortisol and the putative steroid, 18-hydroxy-11-deoxycortisol, was purified by TLC and HPLC and subjected to gas chromatography mass spectrometry. The mass spectra indicated that the steroid isolated was indeed 18-hydroxy-11-deoxycortisol. The function of this steroid is still unknown.     

The synthesis of aldosterone by the adrenal cortex. Two zones (fasciculata and glomerulosa) possess one enzyme for 11 beta-, 18-hydroxylation, and aldehyde synthesis

In order to establish the nature of the aldosterone synthetase activity in the adrenal cortex, we have used porcine adrenal, bovine adrenal cortex, highly purified bovine and porcine 11 beta-/18-hydroxylase, and antibodies raised against the latter enzyme. Mitochondria from two zones (glomerulosa and fasciculata) of the bovine cortex synthesize aldosterone, but those from glomerulosa are much more active than those from fasciculata. Partially purified (cholate-extracted plus ammonium sulfate-precipitated) extracts of mitochondria from the two zones are equally active in catalyzing all three steps in the conversion of 11-deoxycorticosterone to aldosterone. 18-Hydroxylase and aldehyde synthetase activities (18-hydroxycorticosterone----aldosterone) were completely precipitated from cholate extracts of mitochondria from bovine adrenal by antibodies to the pure porcine enzyme. No activity corresponding to any of the three steps in the conversion of 11-deoxycorticosterone to aldosterone was found in extramitochondrial fractions of the bovine cortex. Synthesis of aldosterone by the pure porcine enzyme was inhibited by antibodies to this enzyme and by metyrapone (an inhibitor of 11 beta-/18-hydroxylase). When fractions of porcine adrenal, resulting from purification of the enzyme from mitochondria, were exhaustively tested for any of the enzyme activities required for the synthesis of aldosterone, activity was found only in those fractions containing the 11 beta-/18-hydroxylase, i.e. no additional enzyme was discarded during the purification procedure. It is concluded that the only adrenocortical enzyme capable of synthesizing aldosterone in bovine and porcine adrenal is the well known 11 beta-hydroxylase, that the conversion of 18-hydroxycorticosterone to aldosterone is catalyzed by this cytochrome P-450, and that this step (aldehyde synthetase) requires the heme of the P-450 as demonstrated by the photochemical action spectrum.     

Mutant forms of cytochrome P-450 controlling both 18- and 11beta-steroid hydroxylation in the rat.

A reciprocal relationship between steroid 18- and 11beta-hydroxylase activities in the salt susceptible (S) and the salt resistant (R) strains of rats was previously shown to be controlled by a single genetic locus with two alleles and inheritance by co-dominance (Rapp, J. P., and Dahl, L. K. (1972), Endocrinology 90, 1435). The strain specific steroidogenic patterns, characterized by the relative magnitudes of 18- and 11beta-hydroxylase activities, were found to be determined by adrenal mitochondrial cytochrome P-450 particles. Carbon monoxide inhibition of 18- and 11beta-hydroxylation of deoxycorticosterone in these strains showed that the CO/O2 ratio causing 50% inhibition (i.e., Warburg's partition constant, K) was identical for 18- and 11beta-hydroxylation within a strain, but different for both 18- and 11 beta hydroxylation between strains. (K values were: S rats, 18-hydroxylation = 11.4 +/- 1.4; S rats, 11beta-hydroxylation = 11.0 +/- 1.2; R rats, 18-hydroxylation = 56.4 +/- 13.7; R rats, 11beta-hydroxylation = 46.7 +/- 11.7). This between-strain difference was unique for 18- and 11beta-hydroxylation; i.e., it was not seen with cholesterol side-chain cleavage or 21-hydroxylation. Moreover, the strain-specific K values for 18- and 11beta-hydroxylase and the strain-specific steroidogenic patterns due to the relative magnitudes of 18- and 11beta-hydroxylase activities segregated together in an F2 population. These data strongly suggest the same cytochrome P-450 is involved in both 18- and 11beta-hydroxylation and that this cytochrome is mutated between S and R rats. K values for the reaction corticosterone leads to 18-hydroxycorticosterone were different between S and R strains, indicating that the mutant cytochrome was also involved in this hydroxylation, but K values for the conversion corticosterone leads to aldosterone were not different between strains. This was interpreted to mean that each step in the sequence corticosterone leads to 18-hydroxycorticosterone leads to aldosterone was mediated by a different cytochrome, the K value for the second step being the lower and dominating the overall reaction. It was speculated that the second step could be a second hydroxylation at position 18 to yield 18,18-dihydroxycorticosterone which could be unstable and decompose into aldosterone and water.     

Assay and properties of 18-hydroxylation of endogenous and exogenous corticosterone in rat adrenals. Evidence for heterogeneity of 18-hydroxylase activity.

A mass fragmentographic technique for assay of 18-hydroxylation of labeled (exogenous) and unlabeled (endogenous) corticosterone in adrenal mitochondria and in reconstituted cytochrome P-450 systems has been developed. An extract of an incubation of [14-14C]corticosterone is subjected both to thin-layer radiochromatography and to mass fragmentography (as O-methyloxime-trimethylsilyl ether derivative). In the latter procedure the ions at m/e 605 and 607 (specific for the derivatives of unlabeled and labeled 18-hydroxycorticosterone, respectively), at m/e 591 and 593 (specific for the derivatives of unlabeled labeled aldosterone, respectively) and at m/e 548 and 550 (specific for the derivatives of unlabeled and labeled corticosterone, respectively) were followed through the gas-liquid chromatography. From the ratio between the peaks obtained in the mass fragmentography and from the percentage conversion of [4-14C]corticosterone obtained in the thin-layer radiochromatography, the amount of endogenous and exogenous 18-hydroxycorticosterone and aldosterone could be calculated. The effects of time, enzyme, and substrate concentration of 18-hydroxylation were studied and optimal conditions for assay were determined. Under most conditions, the ratio between labeled and unlabeled 18-hydroxylated products was about constant, indicating that labeled and unlabeled corticosterone were not in equilibrium. It was ascertained that the 18-hydroxycorticosterone and aldosterone formed in the incubations were derived from corticosterone. [4-14C]18-Hydroxydeoxycorticosterone was not converted into aldosterone or 18-hydroxycorticosterone. In vitro studies with different 18-hydroxylase inhibitors (spironolactone, canrenone, and canrenoate-K) and studies with rats pretreated with KCl in drinking fluid suggest that 18-hydroxylation of corticosterone is catalyzed by an enzyme system different from that catalyzing 18-hydroxylation of deoxycorticosterone.     

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.     

Interaction of CYP11B1 (cytochrome P-45011 beta) with CYP11A1 (cytochrome P-450scc) in COS-1 cells.

The interactions of CYP11B1 (cytochrome P-45011beta), CYP11B2 (cytochrome P-450aldo) and CYP11A1 (cytochrome P-450scc) were investigated by cotransfection of their cDNA into COS-1 cells. The effect of CYP11A1 on CYP11B isozymes was examined by studying the conversion of 11-deoxycorticosterone to corticosterone, 18-hydroxycorticosterone and aldosterone. It was shown that when human or bovine CYP11B1 and CYP11A1 were cotransfected they competed for the reducing equivalents from the limiting source contained in COS-1 cells; this resulted in a decrease of the CYP11B activities without changes in the product formation patterns. The competition of human CYP11A1 with human CYP11B1 and CYP11B2 could be diminished with excess expression of bovine adrenodoxin. However, the coexpression of bovine CYP11B1 and CYP11A1 in the presence of adrenodoxin resulted in a stimulation of 11beta-hydroxylation activity of CYP11B1 and in a decrease of the 18-hydroxycorticosterone and aldosterone formation. These results suggest that the interactions of CYP11A1 with CYP11B1 and CYP11B2 do not have an identical regulatory function in human and in bovine adrenal tissue.