11β-hydroxylation Of Deoxycorticosterone Research Articles

Mutations in aldosterone synthase gene of Milan hypertensive rats: phenotypic consequences.

Using in vitro and in vivo methods, we have demonstrated increased sensitivity of adrenocortical steroidogenesis to ACTH in Milan hypertensive (MHS) compared with normotensive (MNS) rats and have investigated whether this is caused by mutations of steroidogenic enzymes. Genes encoding aldosterone synthase (CYP11B2) and 11beta-hydroxylase (CYP11B1) in MHS and MNS have been cloned and sequenced. Nucleotide 752 (G) in exon 4 of MHS CYP11B2 differs from that of MNS (A); CYP11B1 sequences were identical. The nucleotide 752 mutation caused a Q251R substitution in the amino acid sequence of MHS CYP11B2. The phenotype of MHS CYP11B2 alleles, when expressed in COS-1 cells, differed from that of MNS alleles. The relative activities of the three reactions catalyzed by CYP11B2 (11beta-hydroxylation of deoxycorticosterone, 18-hydroxylation of corticosterone, and dehydrogenation of 18-hydroxycorticosterone) were estimated after incubation of transfected cells with [(14)C]deoxycorticosterone and analysis of radioactivity associated with deoxycorticosterone, corticosterone, 18 hydroxycorticosterone, and aldosterone. Both 11- and 18-hydroxylase activities were lower (19 and 12%, respectively; P < 0.01 and P < 0.05) in cells transfected with MHS compared with MNS alleles, whereas 18-oxidase activity was 42% higher (P < 0.01). To assess the significance of the CYP11B2 mutation in vivo, DNA from F2 hybrid MHS x MNS rats was genotyped. MHS alleles were associated with lower urine volumes in both sexes, lower ventricle weights in male rats, but no difference in systolic or diastolic blood pressures between the sexes. We conclude that a mutation in CYP11B2 may affect aldosterone secretion in MHS; however, under normal environmental circumstances, we were unable to demonstrate any influence of this mutation on blood pressure.

Cloning and expression of the rat adrenal cytochrome P-450 11B3 (CYP11B3) enzyme cDNA: Preferential 18-hydroxylation over 11β-hydroxylation of DOC

The biosynthesis of glucocorticoids and mineralocorticoids in the rat adrenal cortex requires the action of two different cytochrome P450 11β-hydroxylases, CYP11B1 and CYP11B2, which are distributed in the zona fasciculata and glomerulosa, respectively. The existence of another cytochrome P450-11β gene, CYP11B3, was recently reported. Although CYP11B3 has similar gene structure and great homology to the CYP11B1 and -B2 genes, the CYP11B3 mRNA was not originally detected by reverse transcription-polymerase chain reaction (RT-PCR) and has only recently been cloned and detected from neonatal rat adrenals. Herein we demonstrate RT-PCR detection of CYP11B3 mRNA expressed in adult rat adrenal and brain tissues. The whole coding region of the CYP11B3 enzyme cDNA was cloned and sequenced. When transiently expressed in COS-7 cells the CYP11B3 converted deoxycorticosterone (DOC) to corticosterone and 18-hydroxydeoxycorticosterone, but not to 18-hydroxycorticosterone or aldosterone. It produced more 18-OH-DOC than corticosterone. A single mutation in CYP11B3 in which Gly-59 was replaced by Ser, reduced the enzymatic activity 5–6-fold. Furthermore, CYP11B3 mRNA expression is greater in neonatal, compared to adult rat adrenal glands.

Purification and characterization of two distinct forms of rat adrenal cytochrome P450 11β: Functional and structural aspects

It is generally accepted that the last three steps of aldosterone biosynthesis are catalyzed by a single enzyme, i.e., cytochrome P450 11β (P450XIB). We have previously reported that rat adrenal mitochondria may be capable of producing two forms of P450 11β which differ in molecular weight (49 and 51 kDa). In the present study we describe the purification, the enzymatic activities, and some structural properties of these two proteins. Using zona fasciculata mitochondria, the 51-kDa protein was purified to electrophoretic homogeneity by means of octyl-Sepharose chromatography. In a reconstituted system the protein catalyzed 18- and 11β-hydroxylation of deoxycorticosterone, but exhibited no 18-hydroxylation or 18-hydroxydehydrogenation of corticosterone. The 49-kDa protein was isolated from zona glomerulosa mitochondria of rats kept on a low-sodium, high-potassium regimen. Using octyl-Sepharose chromatography, it could be separated from the 51-kDa protein. A reconstituted eluate fraction, containing the 49-kDa protein, converted deoxycorticosterone not only to 18-OH-deoxycorticosterone and corticosterone, but also to 18-OH-corticosterone and aldosterone. These findings indicate that the rat adrenal cortex is capable of producing two distinct forms of active cytochrome P450 11β. A structural relationship of the 49- and 51-kDa proteins was indicated by experiments involving limited proteolysis. Thus, digestion with α-chymotrypsin and V8-protease yielded very similar peptide maps for both proteins. During potassium repletion of potassium-deficient rats, the disappearance of the active 51-kDa protein coincided with the appearance of the 49-kDa protein. These results are suggestive of a post-translational processing mechanism converting the 51-kDa protein into the smaller 49-kDa form. However, the 49-kDa protein might also be encoded by a distinct gene, regulated separately depending on the physiological conditions.

11β-Hydroxylation of 18-hydroxy-11-deoxycorticosterone by rat adrenal tissue: Zone specificity and effect of sodium and potassium restriction

The 11β-hydroxylation of substrate amounts of tritiated 18-hydroxy-11-deoxycorticosterone (18-OH-DOC) by incubated separate zones of the rat adrenal cortex was measured and compared with the 11β-hydroxylation of deoxycorticosterone and 11-deoxycortisol and with the conversion of corticosterone to 18-hydroxycorticosterone. In capsular adrenal tissue (“zona glomerulosa”), 18-OH-DOC, deoxycorticosterone and 11-deoxycortisol were converted to 11β-hydroxysteroids to a similar extent. In the decapsulated portions of the adrenal cortex, the 11β-hydroxylation of 18-OH-DOC was substantially lower than the 11β-hydroxylation of the other two steroids. In both portions of the adrenal cortex more 18-hydroxycorticosterone was produced from added 18-OH-DOC than from added corticosterone. An increased conversion of 18-OH-DOC to 18-hydroxycorticosterone by capsular adrenals of sodiumdeficient rats was observed, but only when the precursor steroid was added at a very high concentration. At all substrate concentrations tested, significantly less 18-OH-DOC was converted to its 11β-hydroxy derivative by capsular adrenals of potassium-deficient rats than by tissue of potassium-replete animals. These findings indicate that the 11β-hydroxylation of 18-OH-DOC is a reaction characteristic of the zona glomerulosa and is regulated in response to alterations in the body sodium and potassium status.

Adrenal hydroxylations in genetically obese and hypertensive rats

The separate steps in the formation of aldosterone from cholesterol were studied in a strain of spontaneously hypertensive rats in which phenotypic obesity is inherited as a recessive trait (Koletsky rats). The obese and hypertensive state had little or no effect on side-chain cleavage of cholesterol, formation of progesterone from pregnenolone or 21-hydroxylation. Mitochondrial 18-hydroxylation of endogenous and exogenous corticosterone, however, as well as 18- and 11β-hydroxylation of deoxycorticosterone, were increased in obese hypertensive rats, both when compared with non-obese hypertensive siblings and when compared with healthy Sprague-Dawley rats. 18-Hydroxylation of corticosterone was increased more than 18-hydroxylation of deoxycorticosterone. In non-obese hypertensive rats, the adrenal content of mitochondrial cytochrome P-450 was lower than that in obese hypertensive rats but higher than that in rats of the conventional Sprague-Dawley strain. The results are discussed with respect to possible heterogeneity of adrenal cytochrome P-450 and to possible explanations for the changes observed.

Common characteristics of the cytochrome P-450 system involved in 18- and 11β-hydroxylation of deoxycorticosterone in rat adrenals

18- and 11β-Hydroxylation of deoxycorticosterone and side chain cleavage of cholesterol were studied in mitochondria and submitochondrial reconstituted systems prepared from rat and bovine adrenals. A mass fragmentographic technique was used that allows determination of hydroxylation of both exogenous and endogenous cholesterol. The following results were obtained. (1) Treatment of rats with excess potassium chloride in drinking fluid increased mitochondrial cytochrome P-450 as well as 18- and 11β-hydroxylase activity in the adrenals. Cholesterol side chain cleavage was not affected. In the presence of excess adrenodoxin and adrenodoxin reductase, cytochrome P-450 isolated from potassium chloride-treated rats had higher 18- and 11β-hydroxylase activity per nmol than cytochrome P-450 isolated from control rats. The stimulatory effects on 18- and 11β-hydroxylation were of similar magnitude. (2) Long-term treatment with ACTH increased cholesterol side chain cleavage in the adrenals but had no effect on 18- and 11β-hydroxylase activity. The amount of cytochrome P-450 in the adrenals was not affected by the treatment. It was shown with isolated mitochondrial cytochrome P-450 in the presence of excess adrenodoxin and adrenodoxin reductase that the effect of ACTH was due to increase of side chain cleavage activity per nmol cytochrome P-450. Side chain cleavage of exogenous cholesterol was affected more than that of endogenous cholesterol. (3) Gel chromatography of soluble cytochrome P-450 prepared from rat and bovine adrenal mitochondria yielded chromatographic fractions having either a high 18- and 11β-hydroxylase activity and a low cholesterol side chain cleavage activity or the reverse. The ratio between 18- and 11β-hydroxylase activity was approximately constant, provided the origin of cytochrome P-450 was the same. (4) Addition of progesterone to incubations of deoxycorticosterone with soluble or insoluble rat adrenal cytochrome P-450 competitively inhibited 18- and 11β-hydroxylation of deoxycorticosterone to the same degree. Addition of deoxycorticosterone competitively inhibited 11β-hydroxylation of progesterone with the same system. Progesterone was not 18-hydroxylated by the system. From the results obtained, it is concluded that 18- and 11β-hydroxylation have similar properties and that the binding site for deoxycorticosterone is similar or identical in the two hydroxylations. The possibility that the same specific type of cytochrome P-450 is responsible for both 18- and 11β-hydroxylation of deoxycorticosterone is discussed.

Immunochemical evidence for the involvement of adrenal ferredoxin in the side-chain cleavage of 20α-hydroxycholesterol

A goat antibody produced against homogeneous bovine adrenal ferrodoxin has been employed to study the involvement of this iron-sulfur protein in the side-chain cleavage of 20α-hydroxycholesterol catalyzed by a soluble fraction, supernatant S 1, prepared from sonicated bovine adrenocortical mitochondria. When added to this supernatant, the antibody inhibited the side-chain cleavage of 20α-hydroxycholesterol as well as the side-chain cleavage of cholesterol, the 11β-hydroxylation of deoxycorticosterone, and the NADPH-dependent reduction of cytochrome c . These results demonstrate that, similar to the NADPH-cytochrome c reductase and both the cholesterol side-chain cleavage and steroid 11β-hydroxylase reactions, adrenal ferredoxin is also required for the side-chain cleavage of 20α-hydroxycholesterol.

Inhibition of rat adrenal steroidogenesis by adenine containing compounds

The succinate-supported synthesis of corticosterone from endogenous precursors in a cell-free system from rat adrenals is inhibited by ADP and ATP. The inhibition is observed at short incubation times followed by a recovery from the inhibition and it appears to be specific for adenine containing compounds. The inhibition by ADP(ATP) requires phosphate, is increased by the presence of glucose plus hexokinase and eliminated by the presence of atractyloside or phosphoenolpyruvate plus pyruvate kinase. The inhibition is also eliminated if ATP is added shortly after the start of the incubation or if succinate is replaced by isocitrate. The inhibition has been found to occur only at the 11β-hydroxylation of deoxycorticosterone and the inhibition of this reaction shares many of the properties of the inhibition of corticosterone synthesis from endogenous precursors. It is concluded that the inhibition may be attributed to a competition for reducing equivalents between the hydroxylase system and oxidative phosphorylation as well as a direct effect on the hydroxylase system.

Immunochemical studies on adrenal ferredoxin: Involvement of adrenal ferredoxin in the cholesterol side-chain cleavage reaction of mammalian adrenals

A goat antibody produced against bovine adrenal ferredoxin has been employed to establish immunochemically the involvement of adrenal ferredoxin in the cholesterol side-chain cleavage reaction catalyzed by mammalian adrenal mitochondria. When added to preparations of bovine adrenocortical mitochondria, this antibody was found to inhibit the conversion of cholesterol to pregnenolone and progesterone, the 11β-hydroxylation of deoxycorticosterone and the NADPH-dependent reduction of cytochrome c. These observations demonstrate that, similar to the NADPH-cytochrome c reductase and steroid 11β-hydroxylase reactions, adrenal ferredoxin is also required for the oxidative cleavage of the cholesterol side-chain catalyzed by bovine adrenocortical mitochondria. The goat antibody to bovine adrenal ferredoxin was also found to interact with the comparable iron-sulfur proteins present in mitochondria prepared from sheep, rat, mouse, cat, dog, guinea pig, rabbit, and human adrenals. The interaction of the antibody with these iron-sulfur proteins resulted in the inhibition of both the cholesterol side-chain cleavage and NADPH-cytochrome c reductase activities catalyzed by these adrenal mitochondria. The NADH-dependent reduction of cytochrome c catalyzed by mammalian adrenal mitochondria was not inhibited by the goat antibody to adrenal ferredoxin. These results demonstrate the immunochemical similarity existing among mammalian adrenal ferredoxins and their involvement in the adrenal cholesterol side-chain cleavage reaction.

The effects of glutathione on the 11 beta-hydroxylation of 11-deoxycorticosterone by bovine adrenal cortex mitochondria.

The effects of glutathione on the 11 beta-hydroxylation of 11-deoxycorticosterone by bovine adrenal cortex mitochondria.

Inhibition of ACTH-stimulated steroidogenesis in isolated rat adrenal cells treated with neuraminidase

The possible role of membrane sialic acid in the action of ACTH was investigated in rat adrenal cells. After treatment with neuraminidase, the cells showed a diminished steroidogenic response to ACTH while the response to cyclic AMP and dibutyryl cyclic AMP was unaffected. 11β-hydroxylation of deoxycorticosterone (DOC) was also not impaired. Dose response curves for three ACTH peptides (ACTH 1–39′, ACTH 1–24 and ACTH 1–10) with neuraminidase treated cells suggest that sialic acid residues on the glycoproteins of the plasma membrane may either impart affinity to the plasma membrane for ACTH molecule or facilitate transmission of the signal arising from ACTH-receptor interaction to the catalytic site of adenyl cyclase.

18-Hydroxy-Deoxycorticosterone Secretion in Experimental Hypertension in Rats

Adrenal steroidogenesis was studied in vitro and in vivo in rats which have been specifically bred for susceptibility (S strain) or resistance (R strain) to the hypertensive effects of salt. A marked quantitative difference was found between S and R rats in the adrenal biosynthetic pathways which are subsequent to deoxycorticosterone (DOC). There was a twofold difference in the ability to 18-hydroxylate DOC to form 18-hydroxy-deoxycorticosterone (18OH-DOC) with the greater activity in the S rats. This increment in 18-hydroxylation of DOC in S over R rats was offset by an equal decrement in 11β-hydroxylation of DOC to form corticosterone (B) in S compared with R rats. These alterations in metabolic pathways gave rise to characteristic 18OH-DOC/B ratios for S and R animals that were obvious both in vitro and in vivo. The data suggest, therefore, that in selectively breeding rats on the basis of blood pressure response to salt, a gene (or genes) which affects the 18OH-DOC/B ratio has been segregated in the S and R strains. Although this segregation is in itself good evidence for an effect of 18OH-DOC (or 18OH-DOC/B ratio) on blood pressure in rats, it was also emphasized that there is evidence for more than one gene affecting blood pressure in rats. Thus, the steroid difference described is likely to be controlled by only one of several genes affecting the quantitative character of blood pressure. Comparison of 18OH-DOC/B ratios in S and R strains with other strains of rats showed that the high ratio in S rats was quite common, but that the low ratio in R rats was rather unique. Thus, it seems that the low 18OH-DOC/B ratio of R animals connotes resistance to salt, while the higher ratio in S and unselected strains probably only connotes normal susceptibility to salt. The extreme sensitivity of S rats to salt must therefore be accounted for by other genes.

Further studies on steroidogenesis VIII. Glycolytic breakdown of glucose-6-phosphate and 11β-hydroxylation of 11-deoxycorticosterone in a cell free preparation of the rat adrenal gland

A rat adrenal gland supernatant fraction (representing cytosol of the adrenal cell) fortified with inorganic phosphate, ATP, ADP, NAD +, Mg ++, and HCO 3 − (complete system) metabolized glucose-6-P to lactate and pyruvate under aerobic conditions. Of the two metabolites, lactate was by far the major product formed under most of the incubation conditions. When mitochondria and 11-deoxycorticosterone (DOC) were added to the supernatant fraction, pyruvate was substantially oxidized by the mitochondria but lactate accumulation was increased. Concomitantly, a significant conversion of DOC into corticosterone occurred. When NAD + or Mg ++ were withdrawn from the complete system plus mitochondria both pyruvate and lactate levels were substantially lowered and corticosterone formation was diminished. Omission of inorganic phosphate reduced lactate levels significantly without affecting pyruvate or corticosterone formation. Increasing HCO 3 − concentrations resulted in an almost linear increase in pyruvate formation with relatively little effect on lactate accumulation. In this case, pyruvate production was in excess of that which could be oxidized by the mitochondria, and no further increase in corticosterone formation took place. Pyruvate, but not lactate, was significantly utilized by mitochondria in support of 11β-hydroxylation of DOC. Neither NAD + nor MgCl 2 were required for the utilization of pyruvate by the mitochondria. These observations lead us to conclude that 11β-hydroxylation of DOC was controlled and supported by the pyruvate formed from glucose-6-P in the supernatant fraction and was not related to lactate accumulation per se.

Respiratory activity of adrenal cortex mitochondria during steroid hydroxylation

Mitochondria from the cortex of beef adrenal glands have been isolated and shown to oxidize a variety of tricarboxylic acid cycle substrates concomitant with the 11β-hydroxylation of deoxycorticosterone (DOC). These mitochondria possess a number of activities markedly different from those observed with mitochondria from liver, kidney, heart, etc. Although respiratory control ratios of two to three can be obtained during succinate oxidation, uncouplers or calcium ions stimulate respiration five- to sixfold. The low activity of dinitrophenol-stimulated ATPase suggests a deficiency in energy transfer reactions for ATP formation by adrenal cortex mitochondria favoring the diversion of energy for the support of hydroxylation reactions. This hypothesis is supported by measurements of DOC inhibition of ATP formation during succinate or malate oxidation. The influence of inhibitors on the function of the two types of respiratory chains present in adrenal cortex mitochondria, i.e., a normal cytochrome-containing respiratory chain and a cytochrome P-450-containing respiratory chain, reveals the role of an active malic enzyme. The stoichiometry of DOC hydroxylation demonstrates a ratio of corticosterone: O 2 of 1.1 when malate is used as substrate. Although present studies support a role for an energy-linked transhydrogenase during succinate supported oxidation and DOC hydroxylation, a proposed role for malic enzyme has been considered.

Mechanism of metopirone inhibition of a soluble adrenal steroid 11-beta-hydroxylase.

The effect of Metopirone (2-methyl-1,2-bis(3-pyridyl)-1-propanone) (Su-4885) on the 11β-hydroxylation of 11-deoxycorticosterone (DOC) and NADPH oxidation has been examined in a soluble enzyme system extracted from an acetone powder of bovine adrenal mitochondria. Addition of Metopirone in the absence of DOC stimulated the oxidation of reduced nicotinamide–adenine dinucleotide phosphate (NADPH) by the enzyme system. Under the experimental conditions employed, 11β-hydroxylation was inhibited 50% by Metopirone at a final concentration of 4.2 μmoles/l, a quantity 27 times less than that required to produce a twofold increase in NADPH oxidation in the absence of DOC. Low levels of Metopirone that effectively inhibited corticosterone formation produced a similar decrease in NADPH oxidation associated with the hydroxylation reaction, indicating the interrelationship of 11β-hydroxylation and NADPH oxidation. Evaluation of kinetic data showed that the inhibition of 11β-hydroxylation produced by Metopirone was competitive. The Kmfor the 11β-hydroxylation of DOC was 1.8 × 10−5 mole/l and the Kifor Metopirone was 3.2 × 10−7 mole/l. The ratio Km:Kiindicated that the affinity of Metopirone for the 11β-hydroxylase was 56 times greater than that of DOC.