Chemogenetic activation of adrenocortical Gq signaling causes hyperaldosteronism and disrupts functional zonation.

The mineralocorticoid aldosterone is produced in the adrenal zona glomerulosa (ZG) under the control of the renin-angiotensin II (AngII) system. Primary aldosteronism (PA) results from renin-independent production of aldosterone and is a common cause of hypertension. PA is caused by dysregulated localization of the enzyme aldosterone synthase (Cyp11b2), which is normally restricted to the ZG. Cyp11b2 transcription and aldosterone production are predominantly regulated by AngII activation of the Gq signaling pathway. Here, we report the generation of transgenic mice with Gq-coupled designer receptors exclusively activated by designer drugs (DREADDs) specifically in the adrenal cortex. We show that adrenal-wide ligand activation of Gq DREADD receptors triggered disorganization of adrenal functional zonation, with induction of Cyp11b2 in glucocorticoid-producing zona fasciculata cells. This result was consistent with increased renin-independent aldosterone production and hypertension. All parameters were reversible following termination of DREADD-mediated Gq signaling. These findings demonstrate that Gq signaling is sufficient for adrenocortical aldosterone production and implicate this pathway in the determination of zone-specific steroid production within the adrenal cortex. This transgenic mouse also provides an inducible and reversible model of hyperaldosteronism to investigate PA therapeutics and the mechanisms leading to the damaging effects of aldosterone on the cardiovascular system.

Aldosterone-dependent and -independent regulation of the epithelial sodium channel (ENaC) in mouse distal nephron.

Aldosterone is thought to be the main hormone to stimulate the epithelial sodium channel (ENaC) in the aldosterone-sensitive distal nephron (ASDN) comprising the late distal convoluted tubule (DCT2), the connecting tubule (CNT) and the entire collecting duct (CD). There is immunohistochemical evidence for an axial gradient of ENaC expression along the ASDN with highest expression in the DCT2 and CNT. However, most of our knowledge about renal ENaC function stems from studies in the cortical collecting duct (CCD). Here we investigated ENaC function in the transition zone of DCT2/CNT or CNT/CCD microdissected from mice maintained on different sodium diets to vary plasma aldosterone levels. Single-channel recordings demonstrated amiloride-sensitive Na(+) channels in DCT2/CNT with biophysical properties typical for ENaC previously described in CNT/CCD. In animals maintained on a standard salt diet, the average ENaC-mediated whole cell current (ΔI(ami)) was higher in DCT2/CNT than in CNT/CCD. A low salt diet increased ΔI(ami) in CNT/CCD but had little effect on ΔI(ami) in DCT2/CNT. To investigate whether aldosterone is necessary for ENaC activity in the DCT2/CNT, we used aldosterone synthase knockout (AS(-/-)) mice that lack aldosterone. In CNT/CCD of AS(-/-) mice, ΔI(ami) was lower than that in wild-type (WT) animals and was not stimulated by a low salt diet. In contrast, in DCT2/CNT of AS(-/-) mice, ΔI(ami) was similar to that in DCT2/CNT of WT animals both on a standard and on a low salt diet. We conclude that ENaC function in the DCT2/CNT is largely independent of aldosterone which is in contrast to its known aldosterone sensitivity in CNT/CCD.

Role of genetic variation in regulation of aldosterone biosynthesis.

Aldosterone biosynthesis is not only altered in rare mendelian disorders. Recent evidence suggests that common polymorphisms in the genes mediating the final stages of aldosterone and cortisol production (CYP11B1 and CYP11B2 respectively) are also associated with milder alterations in adrenal corticosteroid biosynthesis. These abnormalities consist of a decrease in adrenal 11ß- hydroxylase activity and a subtle, life-long excess of aldosterone secretion which may lead to long-term cardiovascular risks. An interaction between the CYP11B1 and CYP11B2 genes may exist but is yet to be elucidated. This article describes the studies which highlight the importance of adrenal steroid synthesis in the development of hypertension and cardiovascular dysfunction as well as the role of common polymorphisms in adrenal synthetic genes in altering corticosteroid biosynthesis.

Physiological and pathophysiological applications of sensitive ELISA methods for urinary deoxycorticosterone and corticosterone in rodents.

Deoxycorticosterone (DOC: a weak mineralocorticoid) is the precursor to corticosterone (B: the major glucocorticoid in rodents) and aldosterone (the major mineralocorticoid). The genes Cyp11b1 and Cyp11b2 that encode the enzymes responsible for DOC to B (11beta-hydroxylase) and DOC to aldosterone (aldosterone synthase) conversions are located on the same chromosome. The aim of this study was to develop sensitive and specific ELISA methods to quantify urinary DOC and B concentrations to assess the physiological and genetic control of the Cyp11b1/b2 locus. Antibodies raised in rabbits against DOC and B and horse radish peroxidase-goat anti-rabbit IgG enzyme tracer were used to develop the assays. Urine samples collected from mice held in metabolic cages were extracted with dichloromethane and reconstituted in assay buffer. The assays were validated for specificity, sensitivity, parallelism, accuracy and imprecision. Cross-reactivities with major interfering steroids were minimal: DOC assay (progesterone=0.735% and corticosterone=0.045%), and for B assay (aldosterone=0.14%, 11-dehydro-B=0.006%, cortisol=0.016% and DOC=0.04%) and minimum detection limit for DOC ELISA was 2.2 pg/mL (6.6 pmol/L), and for B ELISA was 6.2 pg/mL (17.9 pmol/L). The validity of urinary DOC and B ELISAs was confirmed by the excellent correlation between the results obtained before and after solvent extraction and HPLC (DOC ELISA: Y=1.092X-0.054, R(2)=0.988; B ELISA: Y=1.047X-0.226, R(2)=0.996). Accuracy studies, parallelism and imprecision data were determined and all found to be satisfactory. The methods were used in a series of metabolic cage studies which demonstrated that (i) females produce more DOC and corticosterone than males; (ii) DOC and corticosterone respond to ACTH treatment but not dietary sodium restriction; (iii) DOC:B ratios in Cyp11b1 null mice were >200-fold greater than wild type.

Effects of genetic variation in the aldosterone synthase (CYP11B2) gene on enzyme function.

OBJECTIVE: Evidence suggests that high levels of aldosterone lead to hypertension and increased risk of cardiovascular disease. Around 15% of patients with essential hypertension have a raised aldosterone to renin ratio (ARR) suggesting that aldosterone production is inappropriately high in relation to its principal agonist angiotensin II. This may be due to increased activity of aldosterone synthase caused by genetic variation in the CYP11B2 gene. We screened the coding region of human CYP11B2 for genetic variants and tested their effects on function in vitro. PROTOCOL: Normotensive subjects (n = 69) were screened for sequence variants in the coding region of CYP11B2 by single-stranded conformation polymorphism (SSCP) analysis and sequencing. The effects of nonsynonymous variants on enzyme activity were assessed in JEG-3 cells transiently transfected with wild-type or variant expression plasmids. The conversion of the substrate 11-deoxycorticosterone (DOC) to corticosterone (B) and aldosterone was measured. RESULTS: Twenty variants were detected in CYP11B2 and eight analysed functionally (Arg87Gly, Asn281Thr, Gly288Ser, Lys296Asn, Asp335Asn, Gln404Arg, Ala414Pro and His439Tyr). Corticosterone synthesis was unaltered and aldosterone synthesis reduced in variant Arg87Gly; Asn281Thr increased corticosterone and decreased aldosterone production; Gly288Ser increased corticosterone production and abolished aldosterone production; Lys296Asn reduced both corticosterone and aldosterone production; Asp335Asn increased corticosterone synthesis but did not affect aldosterone production. Variants Gln404Arg, Ala414Pro and His439Tyr showed increases in both corticosterone and aldosterone synthesis compared to the wild-type. CONCLUSION: The study confirms the genetic variability of the CYP11B2 gene and provides us with additional valuable structure-function information.

Eph receptors and zonation in the rat adrenal cortex.

Although the zonation of the adrenal cortex has a clear functional role, the mechanisms that maintain it remain largely conjectural. The concept that an outer proliferative layer gives rise to cells that migrate inwards, adopting sequentially the zona glomerulosa, fasciculata and reticularis phenotypes, has yet to be explained mechanistically. In other tissues, Eph receptor (EphR)/ephrin signalling provides a mechanism for cellular orientation and migration patterns. Real-time PCR and other methods were used to determine the possible role of Eph/ephrin systems in the rat adrenal. mRNA coding for several members of the EphR family was detected, but out of these, EphA2 provided the closest parallel to zonal organisation. In situ hybridisation showed that EphA2 mRNA and EphA protein were predominantly located in the zona glomerulosa. Its transcription closely reflected expected changes in the glomerulosa phenotype, thus it was increased after a low-sodium diet, but decreased by pretreatment with the angiotensin-converting enzyme inhibitor, captopril. It was also decreased by ACTH treatment, but unaffected by betamethasone. mRNA coding for ephrin A1, the major ligand for the EphA receptors, was also detected in the rat adrenal, though changes evoked by the various pretreatments did not clearly reflect the expected changes in zonal function. Because the maintenance of cellular zonation requires clear positional signals within the adrenal cortex, these data support a role for Eph forward and reverse signalling in the maintenance of adrenocortical zonation.

HindIII(+/-) polymorphism of the Y chromosome, blood pressure, and serum lipids: no evidence of association in three white populations.

BACKGROUND: Male sex is associated with elevated levels of cardiovascular risk factors, including higher blood pressure (BP). Genetic variants on the Y chromosome may contribute to explain the sexual dimorphism in cardiovascular diseases. Among them, the HindIII(+/-) polymorphism of the male-specific region of the Y chromosome has been associated with BP and serum cholesterol levels, with conflicting results. We evaluated the association between the HindIII(+/-) polymorphism, prevalence of hypertension, BP, and serum lipid levels in a large sample of white men and the previously reported epistatic interaction between HindIII(+/-) and the -344C/T polymorphism of the aldosterone synthase gene (CYP11B2) on BP. METHODS: From three European populations (UK n = 422; Belgium n = 313; Italy n = 1248) 1983 white men were phenotyped for BP and serum lipids and genotyped for HindIII(+/-) site and for -344C/T polymorphism in the promoter of CYP11B2 by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). RESULTS: A higher frequency of the HindIII (+) was found in Italians (63%) as compared to both British (31%) and Belgians (28%) (P < .0001). We found no evidence of association of the HindIII(+/-) site with prevalence of hypertension, BP, and serum lipids in any of the three European populations examined and in the entire sample. Finally, we did not observe any interaction between the HindIII(+/-) polymorphism and the -344C/T variant of CYP11B2 on BP. CONCLUSIONS: Our data do not support the hypothesis that the HindIII(+/-) site of the Y chromosome is a marker of cardiovascular risk in white men, highlighting the need for replication in genetic association studies.

Cardiac specific increase in aldosterone production induces coronary dysfunction in aldosterone synthase-transgenic mice.

BACKGROUND: Elevated circulating aldosterone level is associated with impaired cardiovascular function. Although the mechanisms are not fully understood, aldosterone antagonists decrease total and cardiovascular mortality in heart failure and myocardial infarction. Aldosterone induces cardiac fibrosis in experimental models, and it is synthesized locally in rat heart. These observations suggest pathological effects of aldosterone in heart that remain unclear. METHODS AND RESULTS: Transgenic mice (TG) that overexpress the terminal enzyme of aldosterone biosynthesis, aldosterone synthase (AS), in heart have been raised by gene targeting with the alpha-myosin heavy chain promoter. AS mRNA increased 100-fold and aldosterone concentration 1.7-fold in hearts of male TG mice relative to wild-type. No structural or myocardial alterations were evidenced, because ventricle/body weight, AT1 and AT2 receptor binding, and collagen content were unchanged in TG. No alteration in cardiac function was evidenced by echocardiography, isolated perfused heart, or whole-cell patch clamp experiments. In contrast, coronary function was impaired, because basal coronary flow was decreased in isolated perfused heart (-55% of baseline values), and vasodilatation to acetylcholine, bradykinin, and sodium nitroprusside was decreased by 75%, 60%, and 75%, respectively, in TG mice compared with wild-type, showing that the defect was not related to NO production. CONCLUSIONS: Increased cardiac aldosterone production in male mice induces a major coronary endothelium-independent dysfunction with no detectable alterations in cardiac structure and function. However, coronary dysfunction may be harmful for coronary adaptation to increased flow demand.

Studies on the origin of circulating 18-hydroxycortisol and 18-oxocortisol in normal human subjects.

18-Hydroxycortisol (18-OHF) and 18-oxocortisol (18-oxoF) are derivatives of cortisol found in primary aldosteronism but whose origin and regulation in normal subjects are uncertain. 18-OHF can be synthesized by zona fasciculata 11-beta hydroxylase; 18-oxoF can only be produced by zona glomerulosa aldosterone synthase (AS). Stably transfected cell lines expressing either CYP11B1 (11beta-hydroxylase) or CYP11B2 (AS) were incubated with cortisol and other substrates over a range of concentrations. Both enzymes could synthesize 18-OHF from cortisol, but only AS could synthesize 18-oxoF. AS was more efficient than 11beta-hydroxylase at 18-hydroxylation. The apparent Michaelis-Menten constant (K(m)) of AS for cortisol was estimated to be 2.6 microm. In five patients with adrenal insufficiency maintained on hydrocortisone, urinary free cortisol and cortisone levels were high; 18-oxoF was detectable in all patients and 18-OHF in three. It is likely that the 18-oxygenated steroids were synthesized from circulating cortisol, either in the zona glomerulosa or at extraadrenal sites. In eight male volunteers, dexamethasone treatment decreased urinary excretion rates of free cortisol, cortisone, 18-OHF, and 18-oxoF, confirming dependence of 18-oxygenated steroid levels on cortisol availability. In both groups, hydrocortisone administration resulted in detectable levels of 18-OHF and raised levels of 18-oxoF. There was close correlation between 18-oxoF and cortisol excretion during hydrocortisone administration in normal subjects (r = 0.86; P < 0.001). These data show, for the first time, that 18-OHF and 18-oxoF can be synthesized from circulating cortisol. The close correlation between 18-oxoF and cortisol suggests that 18-oxoF is normally produced by the action of AS using circulating cortisol as a substrate. Although 18OHF can be synthesized using circulating cortisol as substrate, our data suggest this is normally produced in the zona fasciculata by 11beta-hydroxylase from locally available cortisol.

Impact of renin-angiotensin-aldosterone system gene variants on the severity of hypertension in patients with newly diagnosed hypertension.

BACKGROUND: The severity of hypertension has prognostic significance. Previous studies have assessed the relationship between renin-angiotensin-aldosterone system (RAAS) genotype and the severity of hypertension in either treated patients or those who have only recently discontinued treatment. METHODS: We assessed the impact of RAAS genotype on ambulatory and office blood pressure (BP) in 231 newly diagnosed hypertensive patients of African ancestry who had never received therapy. Subjects were genotyped for variants of the angiotensin-converting enzyme (insertion/deletion), angiotensinogen (M235T, -20A-->C), and aldosterone synthase (CYP11B2)(-344C-->T) genes. RESULTS: The CYP11B2 gene polymorphism was associated with systolic BP (SBP). In comparison to subjects with at least one copy of the -344C allele (n = 75), patients who were homozygous for the -344T allele (n = 156) had both higher ambulatory SBP (150 +/- 1 v 144 +/- 1 mm Hg, P =.002 before and P =.01 after adjusting for multiple genotyping) and office SBP (163 +/- 2 v 156 +/- 2 mm Hg, P =.01 before and P =.05 after adjusting for multiple genotyping). Neither the angiotensin-converting enzyme insertion/deletion nor the angiotensinogen gene polymorphisms were associated with ambulatory or office SBP or diastolic BP (DBP). The CYP11B2 gene variant also did not affect DBP. CONCLUSION: A variant within the CYP11B2 locus has a clinically important impact on the severity of SBP changes in individuals with newly diagnosed hypertension who are of African ethnicity.

The human steroid hydroxylases CYP1B1 and CYP11B2.

Major advances have been made during the last decade in our understanding of adrenal steroid hormone biosynthesis. Two key players in these pathways are the human mitochondrial cytochrome P450 enzymes CYP11B1 and CYP11B2, which catalyze the final steps in the biosynthesis of cortisol and aldosterone. Using data from mutations found in patients suffering from steroid hormone-related diseases, from mutagenesis studies and from the construction of three-dimensional models of these enzymes, structural information could be deduced that provide a clue to the stereo- and regiospecific steroid hydroxylation reactions carried out by these enzymes. In this review, we summarize the current knowledge on the physiological function and the biochemistry of these enzymes. Furthermore, the pharmacological and toxicological importance of these steroid hydroxylases, the means for the identification of their potential inhibitors and possible biotechnological applications are discussed.

Relationship between transcardiac extraction of aldosterone and left ventricular remodeling in patients with first acute myocardial infarction: extracting aldosterone through the heart promotes ventricular remodeling after acute myocardial infarction.

OBJECTIVES: The purpose of this study was to evaluate whether plasma aldosterone (ALD) is extracted or produced through the heart in patients with acute myocardial infarction (AMI) and to determine the relationship between transcardiac extraction of plasma ALD and left ventricular (LV) remodeling. BACKGROUND: Although we demonstrated that circulating ALD was extracted through the failing heart and that transcardiac extraction of ALD correlated with LV end-diastolic volume index (LVEDVI) in patients with congestive heart failure, the existence and increase of ALD synthase in the hearts of infarct rats were reported, suggesting cardiac production of ALD in patients with AMI. METHODS: We measured plasma ALD in the aortic root (Ao) and coronary sinus (CS) in 57 consecutive patients who received successful revascularization and enalapril, with first AMI at acute phase and after one month. We also measured plasma procollagen type III aminoterminal peptide (PIIINP) in the CS. RESULTS: Plasma ALD was significantly lower in the CS than it was in the Ao at the acute phase (84.7 +/- 6.3 pg/ml vs. 105.5 +/- 8.0 pg/ml, p < 0.0001). Significant positive correlations exist between the transcardiac gradient of ALD at the acute phase and the LVEDVI at one month. Moreover, the transcardiac gradient of plasma ALD at the acute phase has a significant correlation with plasma PIIINP, a biochemical marker of fibrosis, after one month. Stepwise multivariate analysis showed that transcardiac extraction of plasma ALD at the acute phase had an independent and significant positive relationship with a large LVEDVI after one month. CONCLUSIONS: These results indicate that plasma ALD is extracted through the heart in patients with AMI at the acute phase and that the extracted ALD plays an important role in modulating post-infarct LV remodeling.

Amino acid residue 147 of human aldosterone synthase and 11beta-hydroxylase plays a key role in 11beta-hydroxylation.

A number of amino acids differ between aldosterone synthase and 11beta-hydroxylase. To assess their importance in determining the different functional specificities, we substituted aldosterone synthase-specific (aspartate D147, isoleucine I248, glutamine Q43, and threonine T493) with 11beta-hydroxylase-specific amino acids (glutamate E147, threonine T248, arginine R43, and methionine M493), respectively. I248T, Q43R, and T493M had no effect on steroid production compared to wild-type aldosterone synthase. However, CYP11B2-D147E caused a significant increase in corticosterone production and a smaller increase in aldosterone production from 11-deoxycorticosterone (DOC). This appeared to be predominantly due to an increase in the 11beta-hydroxylation of DOC to corticosterone mediated by a decrease in Km, which was 1.4 micromol/L for the mutant compared with 5 micromol/L for the wild-type enzyme. CYP11B2-D147E had no effect on the conversion of 11-deoxycortisol to cortisol. The reverse construct (CYP11B1-E147D), substituting the 11beta-hydroxylase residue with the aldosterone synthase equivalent, decreased the conversion of DOC to corticosterone, which was mediated by an increase in Km that was 7.5 micromol/L for the mutant compared with 2.5 micromol/L for the wild-type enzyme. Again, the conversion of 11-deoxycortisol to cortisol was unimpaired. Thus, amino acid 147 is involved in the transformation of the 17-deoxysubstrate, but not the 17alpha-hydroxysubstrate. The results demonstrate that a conservative change in amino acid, even at some linear distance from known active centers, can significantly affect enzyme substrate affinity and subsequent steroid hormone production.

Baroreflex sensitivity and variants of the renin angiotensin system genes.

OBJECTIVES: Because the renin-angiotensin-aldosterone system (RAS) modifies cardiovascular autonomic regulation, we studied the possible associations between baroreflex sensitivity (BRS) and polymorphism in the RAS genes. BACKGROUND: Wide intersubject variability in BRS is not well explained by cardiovascular risk factors or life style, suggesting a genetic component responsible for the variation of BRS. METHODS: Baroreflex sensitivity as measured from the overshoot phase of the Valsalva maneuver and genetic polymorphisms were examined in a random sample of 161 women and 154 men aged 41 to 61 years and then in an independent random cohort of 29 men and 37 women aged 36 to 37 years. An insertion/deletion (I/D) polymorphism of angiotensin-converting enzyme (ACE), M235T variants of angiotensinogen (AGT) and two diallelic polymorphisms in the gene encoding aldosterone synthase (CYP11B2), one in the promoter (-344C/T) and the other in the second intron, were identified by polymerase chain reaction. RESULTS: In the older population, BRS differed significantly across CYP11B2 genotype groups in women (10.1 +/- 4.5, 8.7 +/- 3.8 and 7.1 +/- 3.2 ms x mm Hg(-1) in genotypes -344TT, CT and CC, respectively, p = 0.003 and 11.1 +/- 4.4, 8.9 +/- 4.1 and 7.5 +/- 3.4 ms x mm Hg(-1) in intron 2 genotypes 1/1, 1/2 and 2/2, respectively, p = 0.002), but not in men. No comparable associations were found for BRS with the I/D polymorphism of ACE or the M235T variant of AGT. In the younger population, BRS was even more strongly related to the CYP11B2 promoter genotype (p = 0.0003). The association was statistically significant both in men (p = 0.015) and in women (p = 0.03). CONCLUSIONS: Common genetic polymorphisms in the aldosterone synthase (CYP11B2) gene is associated with interindividual variation in BRS.

Adrenal zonation: clues from 11beta-hydroxylase and aldosterone synthase.

Aldosterone and cortisol are the major mineralocorticoid and glucocorticoid produced by the human adrenal. Circulating levels of angiotensin II and potassium control the adrenal production of aldosterone, while the production of cortisol is controlled mainly by adrenocorticotropin. The capacity of the adrenal cortex to differentially produce aldosterone and cortisol relies to a large degree on the expression of aldosterone synthase (CYP11B2) and 11beta-hydroxylase (CYP11B1). CYP11B2 catalyzes the final steps in the biosynthesis of aldosterone and is expressed solely in the glomerulosa of the adrenal cortex, while CYP11B1 catalyzes the final steps in the biosynthesis of cortisol and is expressed in the fasciculata/reticularis. The zonal expression of these two isozymes appears to result from transcriptional regulation of the two genes. Herein, the recent progress in defining the cellular mechanisms that regulate transcription of these two isozymes and thus the capacity of the adrenal gland to differentially produce aldosterone and cortisol is discussed.

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.