Steps Of Aldosterone Biosynthesis Research Articles

Angiotensin II-dependent aldosterone production in the adrenal cortex.

The adrenal cortex is responsible for production of adrenal steroid hormones and is anatomically divided into three distinct zones: zona glomerulosa secreting mineralocorticoids (mainly aldosterone), zona fasciculata secreting glucocorticoids (cortisol), and zona reticularis producing androgens. Importantly, due to their high lipophilicity, no adrenal steroid hormone (including aldosterone) is stored in vesicles but rather gets synthesized and secreted instantly upon cell stimulation with specific stimuli. Aldosterone is the most potent mineralocorticoid hormone produced from the adrenal cortex in response to either angiotensin II (AngII) or elevated K+ levels in the blood (hyperkalemia). AngII, being a peptide, cannot cross cell membranes and thus, uses two distinct G protein-coupled receptor (GPCR) types, AngII type 1 receptor (AT1R) and AT2R to exert its effects inside cells. In zona glomerulosa cells, AT1R activation by AngII results in aldosterone synthesis and secretion via two main pathways: (a) Gq/11 proteins that activate phospholipase C ultimately raising intracellular free calcium concentration; and (b) βarrestin1 and -2 (also known as Arrestin-2 and -3, respectively) that elicit sustained extracellular signal-regulated kinase (ERK) activation. Both pathways induce upregulation and acute activation of StAR (steroidogenic acute regulatory) protein, the enzyme that catalyzes the rate-limiting step in aldosterone biosynthesis. This chapter describes these two salient pathways underlying AT1R-induced aldosterone production in zona glomerulosa cells. We also highlight some pharmacologically important notions pertaining to the efficacy of the currently available AT1R antagonists, also known as angiotensin receptor blockers (ARBs) or sartans at suppressing both pathways, i.e., their inverse agonism efficacy at G proteins and βarrestins.

Haplotype association and synergistic effect of human aldosterone synthase (CYP11B2) gene polymorphisms causing susceptibility to essential hypertension in Indian patients

ABSTRACTBackground: Aldosterone synthase (CYP11B2) is a key enzyme involved in the terminal steps of aldosterone biosynthesis. Genetic variability in CYP11B2 gene has been associated with heterogeneous aldosterone production, which can affect sodium homeostasis and thereby regulation of blood pressure. Hence, the present study was aimed to explore the single-locus variations, haplotype and epistasis patterns of CYP11B2 (C-344T, intron-2 gene conversion and Lys173Arg) gene polymorphisms, and the risk contributed by them to the development of essential hypertension (EHT). Methods: A total of 279 hypertensive patients and 200 normotensive controls were enrolled in this study. C-344T and Lys173Arg polymorphisms of CYP11B2 gene were genotyped by PCR-RFLP method and intron-2 gene conversion (IC) polymorphism by allele-specific PCR analysis. Results: Single-locus analysis revealed significant association of CYP11B2 C-344T and Lys173Arg polymorphisms with EHT (p < 0.05). Considering the sexes, Lys173 allele was found to be at risk for hypertension in males (OR 1.40; 95% CI = 1.01–1.96). Unphased haplotype analysis revealed H1 (T-Conv-Lys; p = 0.0017) to have significant risk for EHT, while haplotype H4 (T-Wt-Arg) had a significant protective effect. Multifactor dimensionality reduction (MDR) interaction analysis found the overall best model with C-344T and IC polymorphisms exhibiting strong synergistic effect. Conclusion: The present study revealed a strong synergistic effect of CYP11B2 C-344T and IC polymorphisms causing susceptibility to EHT and haplotype H1 (-344T-Conv-Lys173) as the risk-conferring factor for hypertension predisposition.

Small-Conductance Ca2+-Activated Potassium Channels Negatively Regulate Aldosterone Secretion in Human Adrenocortical Cells.

Aldosterone, which plays a key role in maintaining water and electrolyte balance, is produced by zona glomerulosa cells of the adrenal cortex. Autonomous overproduction of aldosterone from zona glomerulosa cells causes primary hyperaldosteronism. Recent clinical studies have highlighted the pathological role of the KCNJ5 potassium channel in primary hyperaldosteronism. Our objective was to determine whether small-conductance Ca(2+)-activated potassium (SK) channels may also regulate aldosterone secretion in human adrenocortical cells. We found that apamin, the prototypic inhibitor of SK channels, decreased membrane voltage, raised intracellular Ca(2+) and dose dependently increased aldosterone secretion from human adrenocortical H295R cells. By contrast, 1-Ethyl-2-benzimidazolinone, an agonist of SK channels, antagonized apamin's action and decreased aldosterone secretion. Commensurate with an increase in aldosterone production, apamin increased mRNA expression of steroidogenic acute regulatory protein and aldosterone synthase that control the early and late rate-limiting steps in aldosterone biosynthesis, respectively. In addition, apamin increased angiotensin II-stimulated aldosterone secretion, whereas 1-Ethyl-2-benzimidazolinone suppressed both angiotensin II- and high K(+)-stimulated production of aldosterone in H295R cells. These findings were supported by apamin-modulation of basal and angiotensin II-stimulated aldosterone secretion from acutely prepared slices of human adrenals. We conclude that SK channel activity negatively regulates aldosterone secretion in human adrenocortical cells. Genetic association studies are necessary to determine whether mutations in SK channel subtype 2 genes may also drive aldosterone excess in primary hyperaldosteronism.

Identification of aldosterone production sites in primary aldosteronism

It is important to identify the sites of aldosterone biosynthesis in the adrenal glands, especially those involved in primary aldosteronism (PA). CYP11B2 catalyzes the last step of aldosterone biosynthesis but its close similarity to CYP11B1 has made it difficult to generate specific antibodies that distinguish between these two highly homologous cytochromes. We have recently produced specific monoclonal antibodies against CYP11B2. In addition, we have also produced the specific antibodies against 3BHSD 1/2, which is also the pivotal step in aldosterone biosynthesis. In 3BHSD, type 1 is the predominant form in idiopathic hyperaldosteronism (IHA) with diffuse glomerulosa hyperplasia but type 2 dominant in aldosterone producing adenoma (APA). Many of micro APA expressed 3BHSD2 and CYP11B2 with the absence of c17 and CYP11B1, which could explain the cause of hyperaldosteronism despite the small size of the lesions. In addition, many of the adrenals manifesting PA without discernible lesions demonstrated multiple clusters or nodules of cortical cells demonstrating the similar enzyme expression patterns as in micro APA. These results indicated that UMN or unilateral multiple nodules cases resulting in APA are much higher than previously considered.

The discovery of potent inhibitors of aldosterone synthase that exhibit selectivity over 11-β-hydroxylase

Aldosterone, the final component of the renin–angiotensin–aldosterone system, plays an important role in the pathophysiology of hypertension and congestive heart failure. Aldosterone synthase (CYP11B2) catalyzes the last three steps of aldosterone biosynthesis, and as such appears to be a target for the treatment of these disorders. A sulfonamide–imidazole scaffold has proven to be a potent inhibitor of CYP11B2. Furthermore, this scaffold can achieve high levels of selectivity for CYP11B2 over CYP11B1, a key enzyme in the biosynthesis of cortisol.

Differential regulation of aldosterone synthase and 11beta-hydroxylase transcription by steroidogenic factor-1

11beta-Hydroxylase (hCYP11B1) and aldosterone synthase (hCYP11B2) are closely related isozymes with distinct roles in cortisol and aldosterone production respectively. Aldosterone synthase catalyzes the final step in aldosterone biosynthesis and is expressed only in the zona glomerulosa of the normal adrenal. 11beta-Hydroxylase catalyzes the final reaction in the production of cortisol and is expressed at higher levels in the zona fasciculata. The mechanisms causing differential expression of these genes are not well defined. Herein, we demonstrate contrasting roles for the orphan receptor steroidogenic factor-1 (SF-1) in the regulation of human (h) CYP11B1 and hCYP11B2. Human NCI-H295R (H295R) or mouse Y-1 cells were transiently transfected with luciferase reporter constructs containing 5'-flanking regions of hCYP11B1, hCYP11B2, human 17alpha-hydroxylase (hCYP17), human cholesterol side-chain cleavage (hCYP11A1) or mouse (m) cyp11b2 (mcyp11b2). Co-transfection of vectors encoding SF-1 increased expression of hCYP11B1, hCYP11A1 and hCYP17 constructs, but inhibited hCYP11B2 reporter activity. Murine, bovine and human SF-1 were unable to increase transcription of hCYP11B2 in H295R cells. Both hCYP11B2 and mcyp11b2 promoter constructs were inhibited similarly by human SF-1. In mouse Y-1 cells, reporter expression of hCYP11B2 and mcyp11b2 was very low compared with hCYP11B1 constructs, suggesting that this adrenal cell model may not be appropriate for studies of CYP11B2. Electrophoretic mobility shift assay demonstrated that SF-1 interacted with an element from both hCYP11B1 and hCYP11B2. However, mutation of this element, termed Ad4, did not prevent agonist stimulation of hCYP11B2 by angiotensin II or forskolin but blocked activity of hCYP11B1. In some, but not all, reports of genetic linkage analysis, a naturally occurring single nucleotide polymorphism within the Ad4 element of hCYP11B2 (-344C/T) has been associated with cardiovascular disease. Herein, we have demonstrated that this polymorphism influenced binding of SF-1 in electrophoretic mobility shift assays, with the C allele binding SF-1 more strongly than the T allele. However, when hCYP11B2 constructs containing these alleles were transfected into H295R cells, there was no difference in agonist-stimulated expression or the response of either reporter construct to co-expression with human SF-1. Taken together, these data suggest that SF-1 and the Ad4 element are not major regulators of adrenal hCYP11B2 gene expression. Thus far, hCYP11B2 is the first steroid hydroxylase gene which is not positively regulated by SF-1.

Late steps of aldosterone biosynthesis: sheep are not rats.

1. The last three steps of aldosterone biosynthesis have been demonstrated to be catalysed by a single enzyme, referred to as CYP11B (or P450(11) beta) in cow, pig, sheep and bullfrog and as CYP11B2 (or P450aldo) in rat, human, mouse and hamster. 2. The related enzyme CYP11B1 (also referred to as P450(11) beta) in rat, human, mouse and hamster does not have aldosterone synthesis activity, but no such enzyme has been reported in the cow, pig or sheep to date. 3. Exclusive aldosterone secretion in the zona glomerulosa (ZG) of the adrenal cortex in species such as rat, human, mouse and hamster could be ascribed to the restricted distribution of CYP11B2 to the same region in the adrenal cortex. 4. In other species, such as cow, pig and sheep, the CYP11B enzyme is expressed throughout the adrenal cortex and, thus, the exclusive aldosterone biosynthesis in the ZG could not be explained simply by the distribution of the enzyme. 5. We have shown in the sheep that potassium loading and acute sodium depletion stimulate the CYP11B transcript levels, which are not further increased by chronic sodium depletion. 6. The predominant CYP11B in the sheep adrenal cortex catalyses the synthesis of aldosterone from deoxycorticosterone (DOC) in vitro, is expressed throughout the adrenal cortex and the corresponding transcript levels are increased by K+ loading or sodium depletion. In short, as far as the last step of aldosterone biosynthesis is concerned, sheep are different from rats. In the rat, the CYP11B2 transcript or protein is elevated by K+ loading or sodium depletion, but not the CYP11B1 transcript or protein. 7. We propose that during severe sodium deficiency there is a switch in the aldosterone pathway to one preferentially involving 18-OH-DOC and not corticosterone.

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.

Associations between aldosterone synthase gene polymorphism and the adrenocortical function in males.

Two diallelic polymorphisms, one in the transcriptional regulatory region (promoter) and the other in the second intron, have been identified in the aldosterone synthase (CYP11B2) gene encoding aldosterone synthase, the enzyme catalysing the last steps of aldosterone biosynthesis. We have examined the associations between these genetic variations and adrenocortical function in a cohort of Finnish males. A cross-sectional study. Helsinki University Central Hospital, Finland. Ninety-two males aged 30-55 years. Basal adrenocortical function was assessed by measuring urinary excretion of aldosterone and cortisol. Functional activity was determined by responses of several adrenal steroids to dexamethasone suppression followed by ACTH stimulation. Polymerase chain reactions were used to identify the polymorphisms in the CYP11B2 gene. The -344TT genotype group in the CYP11B2 promoter had lower systolic blood pressures (P=0.039), but higher urinary aldosterone excretion (P=0.016), and 11-deoxycortisol responses to ACTH stimulation (P=0.021) than the-344CC genotype group. Urinary aldosterone excretion (P=0.033), 11-deoxycortisol (P=0.026), and aldosterone (P=0.013) responses to ACTH were higher in the intron 2 conversion than the nonconversion genotype groups. Polymorphisms in or near the aldosterone synthase gene are associated with variations in aldosterone and 11-deoxycortisol production in males. This may modulate the activity of the renin-angiotensin system and thereby contribute to blood pressure regulation.

Associations between human aldosterone synthase (CYP11B2) gene polymorphisms and left ventricular size, mass, and function.

Aldosterone has direct and indirect effects on the heart, and genetic variations in aldosterone synthesis could therefore influence cardiac structure and function. Such variations might be associated with polymorphisms in the gene encoding aldosterone synthase (CYP11B2), the enzyme catalyzing the last steps of aldosterone biosynthesis. A Finnish population sample of 84 persons (44 women) aged 36 to 37 years was studied by M-mode and Doppler echocardiography to assess left ventricular size, mass, and function. Subjects were genotyped through the use of the polymerase chain reaction for two diallelic polymorphisms in CYP11B2: one in the transcriptional regulatory region (promoter) and the other in the second intron. In multiple regression analyses, the CYP11B2 promoter genotype predicted statistically significant variations in left ventricular end-diastolic diameter (beta=.40, P<.0001), end-systolic diameter (beta=.33, P=.0009), and mass (beta=.17, P=.023). These effects were independent of potentially confounding factors, including sex, body size, blood pressure, physical activity, smoking, and ethanol consumption. Genotype groups also differed in a measure of left ventricular diastolic function, the heart rate-adjusted atrial filling fraction (P=.018). Increased dietary salt, which is known to predict increased left ventricular mass, had this effect only in association with certain CYP11B2 genotypes (P<.001). Genetic variations in or near the aldosterone synthase (CYP11B2) gene strongly affect left ventricular size and mass in young adults free of clinical heart disease. These polymorphisms may also influence the response of the left ventricle to increases in dietary salt.

Mechanism-based inactivation of bovine cytochrome P-450(11beta) by 18-unsaturated progesterone derivatives.

Two 18-unsaturated progesterone derivatives, 18-vinylprogesterone (18-VP) and 18-ethynylprogesterone (18-EP) have proved to be potent inhibitors of the bovine cytochrome P-450(11beta), the enzyme involved in the last steps of aldosterone biosynthesis [Delorme, C., Piffeteau, A., Viger, A. & Marquet, A. (1995) Eur. J. Biochem. 232, 247-256]. In the present study, we demonstrate that these two compounds exhibit the characteristics of mechanism-based inactivators of this enzyme. Inactivation followed pseudo-first-order and saturation kinetics. The kinetic parameters of inactivation were k(i) = 0.11 min(-1) and Ki = 4 microM for 18-VP, and k(i) = 0.12 min(-1) and 22 microM for 18-EP. Inactivation of P-450(11beta) activity was strictly dependent on the presence of NADPH. Protection by the substrate deoxycorticosterone was observed, demonstrating a selective modification at the substrate-binding site. With radiolabeled 18-VP, inactivation was shown to be irreversible with a stoichiometry of 1.4 mol bound [3H]18-VP/mol inactivated cytochrome P-450(11beta). SDS/PAGE analysis of the [3H]18-VP-inactivated enzyme showed that, under conditions preventing heme dissociation, the P-450(11beta) band was labeled, while no labeling of the apoprotein was observed under denaturating conditions. Furthermore, the loss of catalytic activity could be correlated with the destruction of the P-450 chromophore evaluated by the FeII-CO versus FeII difference spectra. These arguments led us to propose that 18-vinylprogesterone inactivates cytochrome P-450(11beta) by heme destruction rather than by protein modification.

Specific inhibition of the last steps of aldosterone biosynthesis by 18-vinylprogesterone in bovine adrenocortical cells

18-Vinylprogesterone (18-VP), designed for mechanism-based specific inhibition of the last steps of the aldosterone biosynthesis, was used to characterize the mechanism of the 11- and 18-hydroxylase activities of bovine cytochrome P450 11 β . In the present work, its action was studied by observations on a primary culture of bovine adrenocortical cells. First, we investigated the effects of 18-VP on the different enzymatic steps of the biosynthesis of cortisol and aldosterone. The production of cortisol, baseline or hormone-stimulated (ACTH or AII), was inhibited by 18-VP in a dose-dependent manner with a maximal inhibition at 5 μM. Supply of different exogenous substrates to support steroidogenesis revealed an inhibition of the last step of cortisol or corticosterone biosynthesis. We then used specific blockers to measure individual activities and conclude that 11β-hydroxylation was the only enzymatic activity affected. Aldosterone, as well as 18-hydroxycorticosterone, was also measured following addition of corticosterone. The 18-hydroxylation of corticosterone was inhibited by 18-VP, with 50% inhibition occurring at 0.04 μM compared with the 50% inhibition value of 0.3 μM obtained for 11-hydroxylation. Surprisingly, 18-ethynyl-progesterone (18-EP), which has a structure very similar to 18-VP, only weakly inhibits 11β-hydroxylation. The inhibition of aldosterone formation was also much lower with 18-EP than with 18-VP. These studies demonstrate that 18-VP inhibits only the later steps of aldosterone biosynthesis and more specifically 18- than 11-hydroxylation activity.

Inhibition of bovine cytochrome P-450(11 beta) by 18-unsaturated progesterone derivatives.

The last step of aldosterone biosynthesis, an 11 beta-hydroxylation followed by two 18-hydroxylations, are catalyzed, in the bovine system, by the same enzyme, the cytochrome P-450(11 beta) (deoxycorticosterone (DOC)-->corticosterone-->18-hydroxycorticosterone-->aldosterone). The 11 beta- and 18-hydroxylase activities were studied separately with a reconstituted enzymic system, using 11-deoxy[14C]corticosterone and [3H]corticosterone, respectively, as substrates. The inhibition of 11 beta-hydroxylase activity by corticosterone was competitive (Ki = 60 microM) showing that transformation of both substrates occurs at the same site. Double-label/double-substrate experiments, using an equimolar mixture of 11-deoxy[14C]corticosterone and [3H]corticosterone, suggested that 18-hydroxycorticosterone is directly formed from 11-deoxycorticosterone without the intermediate corticosterone leaving the enzyme. Inhibitions by 18-vinylprogesterone and 18-ethynylprogesterone, potent inhibitors of aldosterone biosynthesis [Viger, A., Coustal, S., Pérard, S., Piffeteau, A. & Marquet, A. (1989) J. Steroid Biochem. 33, 119-124], were characterized for both activities (11 beta- and 18-hydroxylase). The value of reversible Ki for the 18-hydroxylation (Ki = 5 microM for 18-vinylprogesterone and 30 microM for 18-ethynylprogesterone) is lower than that for the 11 beta-hydroxylation (30 microM and 100-150 microM, respectively); the former inhibitor is stronger than the latter for both steps. The binding of substrates and inhibitors to the active site was also examined by difference absorption spectroscopy. 18-Vinylprogesterone gave rise to a type I spectrum with a Ks value of 35 microM close to that of progesterone, while 18-ethynylprogesterone showed a reverse type I spectrum with a much higher Ks value (140 microM). Based on these results, a hypothetical model, involving a conformational change of the enzyme for the second step, is proposed.

Inborn errors of aldosterone biosynthesis in humans

Corticosterone methyl oxidase (CMO) type I and type II deficiencies are inborn errors at the penultimate and ultimate steps in the biosynthesis of aldosterone in humans. Recently, steroid 18-hydroxylase (P450 C18), or aldosterone synthase (P450 aldo), was shown to be a multifunctional enzyme catalyzing these two steps of aldosterone biosynthesis, i.e., the conversion of corticosterone to 18-hydroxycorticosterone and the subsequent conversion of 18-hydroxycorticosterone to aldosterone. This observation suggests that CMO I and CMO II deficiencies are derived from two different mutations in the P450 C18 gene ( CYP11B2). To elucidate whether or not this is the case, we performed molecular genetic studies on CYP11B2 of both types of patients. Nucleotide sequence analysis has indicated that the gene of CMO I deficient patients is completely inactivated by a frameshift to form a stop codon due to a 5-bp nucleotide deletion in exon 1. Sequence analysis of CYP11B2 of CMO II deficient patients has revealed two point mutations, CGG → TGG (Arg 181 → Trp) in exon 3 and G TG → G CG (Val 386 → Ala) in exon 7. CYP11B1, the gene for steroid 11β-hydroxylase (P450 11β) which was previously postulated to be the target for CMO II deficiency, is not impaired in these two types of patients. Expression studies using the corresponding mutant cDNAs have shown that CMO I deficient patients are null mutants with a complete lack of P450 C18 whereas CMO II deficient patients are leaky mutants with an altered P450 C18 activity. These results provide molecular genetic evidence to show that CMO I and CMO II deficiencies are caused by two different mutations in the single gene, CYP11B2, coding for P450 C18, which plays a major role in the biosynthesis of aldosterone in humans

A MUTATION IN THE CYP11B2 GENE IN A PATIENT WITH CORTICOSTERONE METHYLOXIDASE II (CMO II) DEFICIENCY

CMO II deficiency is a defect in the final step in aldosterone biosynthesis. The CYP11B2 gene encodes a cytochrome P450 enzyme which catalyzes this 18-oxidation reaction, as well as the preceding 11-hydroxylation and 18-hydroxylation steps in the zona glomerulosa of the adrenal gland. A newborn female, born to consanguineous parents, presented with severe salt-wasting. Laboratory evaluation revealed an increased level of 18-OH-Corticosterone consistent with the diagnosis of CMO II deficiency. Segments of the patient's CYP11B2 gene, as well as that of her parents and a normal control, were amplified by PCR using specific oligonucleotide primers in areas that differed from the 93% homologous CYP11B1 gene. The PCR products were analyzed by Southern blot and revealed a large mutation of the patient's CYP11B2 gene 5′ of exon 6. Downstream of exon 6, there are no other gross mutations. This mutation differs from those previously described in the literature. Further evaluation of this defect may reveal valuable insight into the mechanism of aldosterone biosynthesis.

Molecular genetic studies on the biosynthesis of aldosterone in humans

Corticosterone methyl oxidase Type I (CMO I) and II (CMO II) have been postulated to be the enzymes involved in the final two steps of aldosterone biosynthesis in humans. We have isolated human cDNAs for P450c11 and P450c18 as well as the corresponding genes, CYP11B1 and CYP11B2. Both protein products of these two genes as expressed in COS-7 cells exhibit steroid 11β-hydroxylase activity, but only P450c18, a product of CYP11B2, carried steroid 18-hydroxylase activity to form aldosterone. These results indicate that CYP11B2 encodes CMO, the actual catalytic function of which is retained by P450c18, a multifunctional enzyme. This conclusion is further supported by the finding that the P450c18 gene, CYP11B2, is mutated at several different loci in patients deficient in CMO I or II.

Severe hypoaldosteronism due to corticosterone methyl oxidase type II deficiency in two boys: metabolic and gas chromatography-mass spectrometry studies

Infection-triggered, life-threatening salt-loss and hyperkalaemia developed in two male infants with wasting, inappropriately low plasma aldosterone concentrations and elevated plasma renin activity. The presumptive diagnosis of a defective terminal step in aldosterone biosynthesis was made by the presence of large amounts of 11-dehydrotetrahydrocorticosterone and its 18-hydroxylated metabolite (18-OH-THA), free 18-hydroxycorticosterone (18-OH-B) and 18-hydroxytetrahydrocorticosterone in the urine of both patients. The diagnosis of corticosterone methyl oxidase type II (CMO II) deficiency was confirmed by an elevated urinary 18-OH-THA to tetrahydroaldosterone ratio in one boy and by an elevated plasma 18-OH-B to aldosterone ratio in the other boy. Unknown steroids responsible for the salt-loss were not identified. Sodium supplementation but not short-term high dose oral 9 alpha-fluorcortisol (FF) normalized the hyponatraemia in one patient, in whom sodium (Na+)/potassium (K+) co-transport was decreased. Both patients eventually received long-term FF treatment to prevent impairment of longitudinal growth caused by chronic salt-loss. The diagnosis of CMO II deficiency should always be confirmed by elevated precursor-product ratios in urine or plasma, using radioimmunoassays with prior chromatographic separation. Metabolic studies as the short-term response of serum Na+ to high dose FF may not be helpful in differentiating aldosterone biosynthetic defects from end-organ resistance to mineralocorticoids.

Effects of ACTH on the last step of aldosterone biosynthesis

The production of tritiated aldosterone and tritiated SM (a saponifiable 18-hydroxycorticosterone derivative) by rat adrenals were studied at various incubation times in absence or presence of two concentrations of ACTH. Tritiated 18-hydroxycorticosterone or 18-deoxyaldosterone served as precursors. The lower ACTH concentration (150 pM) increased the production of tritiated aldosterone. Whereas, the higher ACTH concentration (1.5 microM) stimulated tritiated aldosterone production at shorter incubation time (30 min), while after 60 min it inhibited. This time dependency would reflect variations in the levels of endogenous steroids. On the other hand, the effects of ACTH on tritiated SM production were opposite to those on tritiated aldosterone. In effect, while 150 pM ACTH inhibited SM production, 1.5 microM ACTH stimulated it. These results suggest that ACTH promotes opposite effects on the productions of aldosterone and SM and therefore both productions would be coordinated under the regulation of ACTH.

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.

An inherited defect in aldosterone biosynthesis caused by a mutation in or near the gene for steroid 11-hydroxylase.

The final step in aldosterone biosynthesis, an oxidation at position 18 of 18-hydroxycorticosterone, is catalyzed by an enzymatic activity termed corticosterone methyl oxidase II (CMO II). This activity is mediated in vitro by P450c11 (steroid 11-hydroxylase), a cytochrome P-450 enzyme that also catalyzes the preceding two steps of 11-hydroxylation and 18-hydroxylation. CMO II deficiency, an inherited defect in the 18-oxidation step, impairs aldosterone biosynthesis and thus leads to a clinical syndrome of salt wasting. To test the hypothesis that CMO II deficiency results from a mutation affecting the structural gene for P450c11, we examined 11 affected and 21 unaffected members of six families with this disorder. After DNA samples were digested with the restriction endonuclease MspI (thereby cutting the DNA at specific sites) and hybridized with a P450c11 DNA probe, a unique DNA fragment in the P450c11 structural gene was detected in subjects with the deficiency. The DNA fragment and the disease trait were inherited together in each family, demonstrating that CMO II deficiency is caused by a mutation in or very near the structural gene for P450c11 on chromosome 8. We conclude that the metabolic diseases of CMO II and 11-hydroxylase deficiency, which have distinct clinical symptoms, may be caused by different mutations in the single gene for a multifunctional enzyme.