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Human cytochrome P450 (P450) 11B2 catalyzes the formation of aldosterone, the major endogenous human mineralocorticoid. Aldosterone is important for the regulation of electrolyte homeostasis. Mutations and overexpression of P450 11B2 (also known as aldosterone synthase) can lead to hypertension, congestive heart failure, and diabetic nephropathy. The enzyme is therefore a target for drug development to manage these various disorders. P450 11B2 catalyzes aldosterone formation from 11-deoxycorticosterone through three distinct oxidation steps. It is currently unknown to which degree these reactions happen in sequence without the intermediate products dissociating from the enzyme ( processively) or whether these reactions happen solely distributively, in which the intermediate products must first dissociate and then rebind to the enzyme before subsequent oxidation. We present here a comprehensive investigation of processivity in P450 11B2-catalyzed reactions using steady-state, pre-steady-state, pulse-chase, equilibrium-binding titrations, and stopped-flow binding studies. We utilized the data obtained to develop a kinetic model for P450 11B2 and tested this model by enzyme kinetics simulations. We found that although aldosterone is produced processively, the enzyme preferentially utilizes a distributive mechanism that ends with the production of 18-OH corticosterone. This seemingly contradictory observation could be resolved by considering the ability of the intermediate product 18-OH corticosterone to exist as a lactol form, with the equilibrium favoring the ring-closed lactol configuration. In summary, our refined model for P450 11B2 catalysis indicates isomerization of the intermediate to a lactol can explain why P450 11B2 must produce aldosterone through a processive mechanism despite favoring a distributive mechanism.
© 2019 Reddish and Guengerich.
The steroid hormone aldosterone regulates sodium and potassium homeostasis. Aldosterone and activation of the mineralocorticoid receptor also causes inflammation and fibrosis of the heart, fibrosis and remodelling of blood vessels and tubulointerstitial fibrosis and glomerular injury in the kidney. Aldosterone and mineralocorticoid-receptor activation initiate an inflammatory response by increasing the generation of reactive oxygen species by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and mitochondria. High salt intake potentiates these effects, in part by activating the Rho family member Rac1, a regulatory subunit of reduced NADPH oxidase that activates the mineralocorticoid receptor. Studies in mice in which the mineralocorticoid receptor has been deleted from specific cell types suggest a key role for macrophages in promoting inflammation and fibrosis. Aldosterone can exert mineralocorticoid-receptor-independent effects via the angiotensin II receptor and via G-protein-coupled receptor 30. Mineralocorticoid-receptor antagonists are associated with decreased mortality in patients with heart disease and show promise in patients with kidney injury, but can elevate serum potassium concentration. Studies in rodents genetically deficient in aldosterone synthase or treated with a pharmacological aldosterone-synthase inhibitor are providing insight into the relative contribution of aldosterone compared with the contribution of mineralocorticoid-receptor activation in inflammation, fibrosis, and injury. Aldosterone-synthase inhibitors are under development in humans.
Angiotensin II causes cardiovascular injury in part by aldosterone-induced mineralocorticoid receptor activation, and it can also activate the mineralocorticoid receptor in the absence of aldosterone in vitro. Here we tested whether endogenous aldosterone contributes to angiotensin II/salt-induced cardiac, vascular, and renal injury by the mineralocorticoid receptor. Aldosterone synthase knockout mice and wild-type littermates were treated with angiotensin II or vehicle plus the mineralocorticoid receptor antagonist spironolactone or regular diet while drinking 0.9% saline. Angiotensin II/salt caused hypertension in both the knockout and wild-type mice, an effect significantly blunted in the knockout mice. Either genetic aldosterone deficiency or mineralocorticoid receptor antagonism reduced cardiac hypertrophy, aortic remodeling, and albuminuria, as well as cardiac, aortic, and renal plasminogen activator inhibitor-1 mRNA expression during angiotensin II treatment. Mineralocorticoid receptor antagonism reduced angiotensin II/salt-induced glomerular hypertrophy, but aldosterone deficiency did not. Combined mineralocorticoid receptor antagonism and aldosterone deficiency reduced blood urea nitrogen and restored nephrin immunoreactivity. Angiotensin II/salt also promoted glomerular injury through the mineralocorticoid receptor in the absence of aldosterone. Thus, mineralocorticoid antagonism may have protective effects in the kidney beyond aldosterone synthase inhibition.
AIMS/HYPOTHESIS - Aldosterone concentrations increase in obesity and predict the onset of diabetes. We investigated the effects of aldosterone on glucose homeostasis and insulin secretion in vivo and in vitro.
METHODS - We assessed insulin sensitivity and insulin secretion in aldosterone synthase-deficient (As [also known as Cyp11b2](-/-)) and wild-type mice using euglycaemic-hyperinsulinaemic and hyperglycaemic clamps, respectively. We also conducted studies during high sodium intake to normalise renin activity and potassium concentration in As (-/-) mice. We subsequently assessed the effect of aldosterone on insulin secretion in vitro in the presence or absence of mineralocorticoid receptor antagonists in isolated C57BL/6J islets and in the MIN6 beta cell line.
RESULTS - Fasting glucose concentrations were reduced in As (-/-) mice compared with wild-type. During hyperglycaemic clamps, insulin and C-peptide concentrations increased to a greater extent in As (-/-) than in wild-type mice. This was not attributable to differences in potassium or angiotensin II, as glucose-stimulated insulin secretion was enhanced in As (-/-) mice even during high sodium intake. There was no difference in insulin sensitivity between As (-/-) and wild-type mice in euglycaemic-hyperinsulinaemic clamp studies. In islet and MIN6 beta cell studies, aldosterone inhibited glucose- and isobutylmethylxanthine-stimulated insulin secretion, an effect that was not blocked by mineralocorticoid receptor antagonism, but was prevented by the superoxide dismutase mimetic tempol.
CONCLUSIONS/INTERPRETATION - We demonstrated that aldosterone deficiency or excess modulates insulin secretion in vivo and in vitro via reactive oxygen species and in a manner that is independent of mineralocorticoid receptors. These findings provide insight into the mechanism of glucose intolerance in conditions of relative aldosterone excess.
Sodium wasting during the neonatal period is the consequence of a physiological aldosterone resistance, related to a low renal mineralocorticoid receptor (MR) expression at birth, both in humans and mice. To investigate whether aldosterone is involved in the neonatal regulation of MR expression, we compared aldosterone and corticosterone levels and renal MR expression by quantitative real-time PCR, between aldosterone synthase (AS) knockout, heterozygous, and wild type (WT) mice, at birth and postnatal d 8. Analysis of MR transcripts showed a similar expression profile in all genotypes, demonstrating that the lack of aldosterone does not modify either the low renal MR expression at birth or its postnatal induction. However, mRNA levels of the α-subunit of the epithelial sodium channel, a MR target gene, were significantly higher in WT compared with AS knockout mice, both at birth and postnatal d 8, despite high corticosterone levels in AS knockout mice, indicating that aldosterone is required for optimal renal induction of the epithelial sodium channel. Using organotypic cultures of newborn WT kidneys, we confirmed that aldosterone does not regulate MR expression at birth, but is instead capable of increasing MR expression in mature kidneys, unlike dexamethasone. In sum, we demonstrate both in vivo and in vitro, that, whereas aldosterone has no significant impact on renal MR expression at birth, it is crucial for optimal MR regulation in postnatal kidneys and for appropriate hydroelectrolytic balance. Understanding of MR-regulatory mechanisms could therefore lead to new therapeutic strategies for the management of sodium loss in preterms and neonates.
PURPOSE OF REVIEW - Aldosterone causes tissue inflammation leading to fibrosis and remodeling in the heart, vasculature, and kidney. We summarize recent data regarding the mechanism(s) through which aldosterone stimulates inflammation.
RECENT FINDINGS - Studies elucidate the cell-specific effects of mineralocorticoid receptor activation on inflammatory cell infiltration and adhesion, and highlight the role of the macrophage in the development of vascular collagen deposition and hypertension. Activation of nuclear factor-kappaB in vascular smooth muscle cells involves a complex interplay between the angiotensin subtype 1 (AT1) receptor and the mineralocorticoid receptor. Activation of the mineralocorticoid receptor by aldosterone stimulates an inflammatory phenotype in adipocytes and contributes to insulin resistance by increasing oxidative stress.
SUMMARY - Mechanistic studies of aldosterone-induced inflammation provide the rationale for an expanded therapeutic role for mineralocorticoid receptor antagonists and aldosterone synthase inhibitors.
In the setting of high salt intake, aldosterone stimulates fibrosis in the heart, great vessels, and kidney of rats. We used uninephrectomized rats treated with angiotensin II and placed on a high salt diet to exaggerate renal fibrosis. We then tested whether mineralocorticoid receptor blockade by spironolactone or aldosterone synthase inhibition by FAD286 have similar effects on end-organ damage and gene expression. Individually, both drugs prevented the hypertensive response to uninephrectomy and high salt intake but not when angiotensin II was administered. Following 4 weeks of treatment with FAD286, plasma aldosterone was reduced, whereas spironolactone increased aldosterone at 8 weeks of treatment. Angiotensin II and high salt treatment caused albuminuria, azotemia, renovascular hypertrophy, glomerular injury, increased plasminogen activator inhibitor-1 (PAI-1), and osteopontin mRNA expression, as well as tubulointerstitial fibrosis in the kidney. Both drugs prevented these renal effects and attenuated cardiac and aortic medial hypertrophy while reducing osteopontin and transforming growth factor-beta mRNA expression in the aorta. The two drugs also reduced cardiac interstitial fibrosis but had no effect on that of the perivascular region. Although spironolactone enhanced angiotensin II and salt-stimulated PAI-1 mRNA expression in aorta and heart, spironolactone and FAD286 prevented renal PAI-1 mRNA protein expression. Our study shows that mineralocorticoid receptor antagonism and aldosterone synthase inhibition similarly decrease hypertrophy and interstitial fibrosis of the kidney and heart caused by angiotensin II and high salt.
To test the hypothesis that angiotensin (Ang) II induces profibrotic gene expression through endogenous aldosterone, we measured the effect of 4 h infusion (600 ng/kg x min) of Ang II on tissue mRNA expression of plasminogen activator inhibitor 1 (PAI-1), preproendothelin-1 (ppET-1), TGF-beta, and osteopontin in wild-type (WT), aldosterone synthase-deficient (AS(-/-)), and AS(-/-) mice treated with aldosterone (either 500 ng/d for 7 d or 250 ng as a concurrent 4 h infusion). Ang II increased aldosterone in WT (P < 0.001) but not in AS(-/-) mice. Aldosterone (7 d) normalized basal aldosterone concentrations in AS(-/-) mice; however, there was no further effect of Ang II on aldosterone (P = NS). Basal cardiac and aortic PAI-1 and ppET-1 expression were similar in WT and AS(-/-) mice. Ang II-stimulated PAI-1 (P < 0.001) and ppET-1 expression (P = 0.01) was diminished in the heart of AS(-/-) mice; treatment with aldosterone for 4 h or 7 d restored PAI-1 and ppET-1 mRNA responsiveness to Ang II in the heart. Ang II increased PAI-1 (P = 0.01) expression in the aorta of AS(-/-) as well as WT mice. In the kidney, basal PAI-1, ppET-1, and TGF-beta mRNA expression was increased in AS(-/-) compared with WT mice and correlated with plasma renin activity. Ang II did not stimulate osteopontin or TGF-beta expression in the heart or kidney. Endogenous aldosterone contributes to the acute stimulatory effect of Ang II on PAI-1 and ppET-1 mRNA expression in the heart; renin activity correlates with basal profibrotic gene expression in the kidney.
The human 11beta-hydroxylase (hCYP11B1) is responsible for the conversion of 11-deoxycortisol into the major mammalian glucocorticoid, cortisol. The reduction equivalents needed for this reaction are provided via a short electron transfer chain consisting of a [2Fe-2S] ferredoxin and a FAD-containing reductase. On the biochemical and biophysical level, little is known about hCYP11B1 because it is very unstable for analyses performed in vitro. This instability is also the reason why it has not been possible to stably express it so far in Escherichia coli and subsequently purify it. In the present study, we report on the successful and reproducible purification of recombinant hCYP11B1 coexpressed with molecular chaperones GroES/GroEL in E. coli. The protein was highly purified to apparent homogeneity, as observed by SDS/PAGE. Upon mass spectrometry, the mass-to-charge ratio (m/z) of the protein was estimated to be 55 761, which is consistent with the value 55 760.76 calculated for the form lacking the translational initiator Met. The functionality of hCYP11B1 was analyzed using different methods (substrate conversion assays, stopped-flow, Biacore). The results clearly demonstrate that the enzyme is capable of hydroxylating its substrates at position 11-beta. Moreover, the determined NADPH coupling percentage for the hCYP11B1 catalyzed reactions using either 11-deoxycortisol or 11-deoxycorticosterone as substrates was approximately 75% in both cases. Biacore and stopped-flow measurements indicate that hCYP11B1 possesses more than one binding site for its redox partner adrenodoxin, possibly resulting in the formation of more than one productive complexes. In addition, we performed CD measurements to obtain information about the structure of hCYP11B1.
Dahl's salt-resistant normotensive rats (DR rats) have been previously reported to express cytochrome P-450 (CYP11B1) containing five missense mutations [Matsukawa, N., Nonaka, Y., Higaki, J., Nagano, M., Mikami H., Ogihara, T. & Okamoto, M. (1993) J. Biol. Chem. 268, 9117-9121]. To investigate structure-function relationships of CYP11B, wild-type rat CYP11B1 and CYP11B2 and DR-CYP11B1 (mutant CYP11B1 in Dahl's salt-resistant rats) have been successfully expressed in Escherichia coli. Steroid 11beta-hydroxylase (11beta-OHase) activity observed with DR-CYP11B1 was similar to that of wild-type CYP11B1, while 18-hydroxylase (18-OHase) activity of DR-CYP11B1 was lower than that of wild-type CYP11B1. Mutant CYP11B1s containing a single or a double amino acid substitution associated with DR-CYP11B1 have been also expressed in E. coli to investigate effects of the substitutions on enzymatic activity. Each of the single mutant enzymes showed lower 18-OHase activity than wild-type CYP11B1, but not as low as DR-CYP11B1. A double mutant CYP11B1 with V381L and I384L showed 18-OHase activity at a similar low level to that of DR-CYP11B1. The 19-hydroxylation (19-OHase) activity of DR-CYP11B1 was about one-third of that of the wild-type enzyme and this low activity appeared due to the V443M mutation. These results suggest that three of five amino acid substitutions present in DR-CYP11B1 account for the decreased 18-OHase and 19-OHase activities. A decrease in these enzyme activities may be responsible for the normotension of the DR rats when fed a high-salt diet.