ATP1A1 Mutant in Aldosterone-Producing Adenoma Leads to Cell Proliferation.

The molecular mechanisms by which ATP1A1 mutation-mediated cell proliferation or tumorigenesis in aldosterone-producing adenomas (APAs) have not been elucidated. First, we investigated whether the APA-associated ATP1A1 L104R mutation stimulated cell proliferation. Second, we aimed to clarify the molecular mechanisms by which the ATP1A1 mutation-mediated cell proliferated. We performed transcriptome analysis in APAs with ATP1A1 mutation. ATP1A1 L104R mutation were modulated in human adrenocortical carcinoma (HAC15) cells (ATP1A1-mutant cells), and we evaluated cell proliferation and molecular signaling events. Transcriptome and immunohistochemical analysis showed that Na/K-ATPase (NKA) expressions in ATP1A1 mutated APA were more abundant than those in non-functioning adrenocortical adenoma or KCNJ5 mutated APAs. The significant increase of number of cells, amount of DNA and S-phase population were shown in ATP1A1-mutant cells. Fluo-4 in ATP1A1-mutant cells were significantly increased. Low concentration of ouabain stimulated cell proliferation in ATP1A1-mutant cells. ATP1A1-mutant cells induced Src phosphorylation, and low concentration of ouabain supplementation showed further Src phosphorylation. We demonstrated that NKAs were highly expressed in ATP1A1 mutant APA, and the mutant stimulated cell proliferation and Src phosphorylation in ATP1A1-mutant cells. NKA stimulations would be a risk factor for the progression and development to an ATP1A1 mutant APA.

The establishment of a novel high-throughput screening system using RNA-guided genome editing to identify chemicals that suppress aldosterone synthase expression.

Aldosterone is synthesized in the adrenal by the aldosterone synthase CYP11B2. Although the control of CYP11B2 expression is important to maintain the mineral homeostasis, its overexpression induced by the depolarization-induced calcium (Ca2+) signaling activation has been reported to increase the synthesis of aldosterone in primary aldosteronism (PA). The drug against PA focused on the suppression of CYP11B2 expression has not yet been developed, since the molecular mechanism of CYP11B2 transcriptional regulation activated via Ca2+ signaling remains unclear. To address the issue, we attempted to reveal the mechanism of the transcriptional regulation of CYP11B2 using chemical screening. We generated a cell line by inserting Nanoluc gene as a reporter into CYP11B2 locus in H295R adrenocortical cells using the CRSPR/Cas9 system, and established the high-throughput screening system using the cell line. We then identified 9 compounds that inhibited the CYP11B2 expression induced by potassium-mediated depolarization from the validated compound library (3399 compounds). Particularly, tacrolimus, an inhibitor of phosphatase calcineurin, strongly suppressed the CYP11B2 expression even at 10 nM. These results suggest that the system is effective in identifying drugs that suppress the depolarization-induced CYP11B2 expression. Our screening system may therefore be a useful tool for the development of novel medicines against PA.

Development of a high-throughput assay for aldosterone synthase inhibitors using high-performance liquid chromatography-tandem mass spectrometry.

Aldosterone plays a key role in the pathogenesis of hypertension, congestive heart failure, and chronic kidney disease. Aldosterone biosynthesis involves three membrane-bound enzymes: aldosterone synthase, adrenodoxin, and adrenodoxin reductase. Here, we report the development of a mass spectrometry-based high-throughput whole cell-based assay for aldosterone synthesis. A human adrenal carcinoma cell line (H295R) overexpressing human aldosterone synthase cDNA was established. The production of aldosterone in these cells was initiated with the addition of 11-deoxycorticosterone, the immediate substrate of aldosterone synthase. An automatic liquid handler was used to gently distribute cells uniformly to well plates. The adaption of a second automated liquid handling system to extract aldosterone from the cell culture medium into organic solvent enabled the development of 96- and 384-well plate formats for this cellular assay. A high-performance liquid chromatography-tandem mass spectrometry method was established for the detection of aldosterone. Production of aldosterone was linear with time and saturable with increasing substrate concentration. The assay was highly reproducible with an overall average Z' value=0.49. This high-throughput assay would enable high-throughput screening for inhibitors of aldosterone biosynthesis.

Angiotensin II receptor blockers differentially affect CYP11B2 expression in human adrenal H295R cells.

We generated a stable H295R cell line expressing aldosterone synthase gene (CYP11B2) promoter/luciferase chimeric reporter construct that is highly sensitive to angiotensin II (AII) and potassium, and defined AII receptor blocker (ARB) effects. In the presence of AII, all ARBs suppressed AII-induced CYP11B2 transcription. However, telmisartan alone increased CYP11B2 transcription in the absence of AII. Telmisartan dose-dependently increased CYP11B2 transcription/mRNA expression and aldosterone secretion. Experiments using CYP11B2 promoter mutants indicated that the Ad5 element was responsible. Among transcription factors involved in the element, telmisartan significantly induced NGFIB/NURR1 expression. KN-93, a CaMK inhibitor, abrogated the telmisartan-mediated increase of CYP11B2 transcription/mRNA expression and NURR1 mRNA expression, but not NGFIB mRNA expression. NURR1 over-expression significantly augmented the telmisartan-mediated CYP11B2 transcription, while high-dose olmesartan did not affect it. Taken together, telmisartan may stimulate CYP11B2 transcription via NGFIB and the CaMK-mediated induction of NURR1 that activates the Ad5 element, independent of AII type 1 receptor.

Disruption of reproductive development in male rat offspring following gestational and lactational exposure to di-(2-ethylhexyl) phthalate and genistein.

Studies of developmental effects of mixtures of endocrine disrupters on the male reproductive system are of great concern. In this study, the reproductive effects of the co-administration of di-2-(ethylhexyl) phthalate (DEHP) and genistein (GEN) during pregnancy and lactation were studied in male rat offspring. Pregnant Sprague-Dawley rats were gavaged from gestation day 3 to postnatal day 21 with vehicle control, DEHP 250 mg/kg body weight (bwyday, GEN 50 mg/kg bwday, GEN 400 mg/kg bwday, and two combinations of the two compounds (DEHP 250 mg/kg bwday + GEN 50 mg/kg bwday, DEHP 250 mg/kg bwday + GEN 400 mg/kg bwday). The outcomes studied were general morphometry (weight, AGD), testicular histology, testosterone levels, and expression at the mRNA level of genes involved in steroidogenesis. Organ coefficient, AGD / body weight1/3 י, serum testosterone concentration and genes involved in steroidogenic pathway expression when exposed to DEHP (250mg/kg bwday), GEN(50mg/kg bwday) or GEN(400mg/kg bwday) alone were not significantly different from the control group. When exposed to (DEHP 250mg/kg bwday +GEN 50mg/kg bwday) together during pregnancy and lactation, serum testosterone concentration, epididymis coefficient and Cypal17a1,Scarb1 m RNA expression significantly decreased compared to the control and GEN(50mg/kg bwday). When exposed to (DEHP 250mg/kg bwday +GEN 400mg/kg bwday) together during pregnancy and lactation, AGD / body weight1/3 י, serum testosterone concentration, testis and epididymis coefficient and Star, Cypal17a1 mRNA expression appeared significantly decreased compared to the control and DEHP/GEN single exposure, together with developmental impairment of seminiferous tubules and seminiferous epithelium. Overall, co-administration of DEHP and GEN during gestation and lactation seem to acts in a cumulative manner to induce the most significant alterations in the neonate, especially with GEN at high dose, although the effect of the DEHP-GEN mixture on adult offspring should be observed further.

Disruption of reproductive development in male rat offspring following gestational and lactational exposure to di-(2-ethylhexyl) phthalate and genistein

Studies of developmental effects of mixtures of endocrine disrupters on the male reproductive system are of great concern. In this study, the reproductive effects of the co-administration of di-2-(ethylhexyl) phthalate (DEHP) and genistein (GEN) during pregnancy and lactation were studied in male rat offspring. Pregnant Sprague-Dawley rats were gavaged from gestation day 3 to postnatal day 21 with vehicle control, DEHP 250 mg/kg body weight (bwyday, GEN 50 mg/kg bwday, GEN 400 mg/kg bwday, and two combinations of the two compounds (DEHP 250 mg/kg bwday + GEN 50 mg/kg bwday, DEHP 250 mg/kg bwday + GEN 400 mg/kg bwday). The outcomes studied were general morphometry (weight, AGD), testicular histology, testosterone levels, and expression at the mRNA level of genes involved in steroidogenesis. Organ coefficient, AGD / body weight1/3 י, serum testosterone concentration and genes involved in steroidogenic pathway expression when exposed to DEHP (250mg/kg bwday), GEN(50mg/kg bwday) or GEN(400mg/kg bwday) alone were not significantly different from the control group. When exposed to (DEHP 250mg/kg bwday +GEN 50mg/kg bwday) together during pregnancy and lactation, serum testosterone concentration, epididymis coefficient and Cypal17a1,Scarb1 m RNA expression significantly decreased compared to the control and GEN(50mg/kg bwday). When exposed to (DEHP 250mg/kg bwday +GEN 400mg/kg bwday) together during pregnancy and lactation, AGD / body weight1/3 י, serum testosterone concentration, testis and epididymis coefficient and Star, Cypal17a1 mRNA expression appeared significantly decreased compared to the control and DEHP/GEN single exposure, together with developmental impairment of seminiferous tubules and seminiferous epithelium. Overall, co-administration of DEHP and GEN during gestation and lactation seem to acts in a cumulative manner to induce the most significant alterations in the neonate, especially with GEN at high dose, although the effect of the DEHP-GEN mixture on adult offspring should be observed further.

[The influence of suoquan capsule on the mRNA expression of CYP11B2 in deficiency of the kidney and diuresis rats].

OBJECTIVE: To explore the mechanism of nourishing kidney and reducing urine effect of suoquan capsule by discussing the adjusting action on ALD synthase. METHODS: Built the model of deficiency of the kidney and diuresis by adenine,inspectd the contents of Cort, ALD in blood and the mRNA expression of CYP11B2 with ELISA and RT-PCR technology of the mice. RESULTS: Compared with model group, Suoquan capsule could remarkedly increase the contents of Cort, ALD in blood and the mRNA expression of CYP1 1 B2 of model rats. CONCLUSIONS: Suoquan capsule can increase the contents of Cort, ALD in blood and the mRNA expression of CYP11B2 in deficiency of the kidney and diuresis rats. Promote the combining of ALD by adjusting the ALD synthase may be the mechanism of nourishing the kidney and reducing urine of Souquan capsule from ALD synthase approach.

The development of a whole-cell based medium throughput screening system for the discovery of human aldosterone synthase (CYP11B2) inhibitors: old drugs disclose new applications for the therapy of congestive heart failure, myocardial fibrosis and hypertension.

Cytochrome P450 enzymes play an important role in steroid hormone biosynthesis of the human adrenal gland, e.g., the production of cortisol and aldosterone. Aldosterone, the most important human mineralocorticoid, is involved in the regulation of the salt and water homeostasis of the body and thus in the regulation of blood pressure, whereas cortisol is the most important glucocorticoid of the human body. CYP11B-dependent steroid hydroxylases are drug development targets, and since they are very closely related enzymes, the discovery of selective inhibitors has been subject to intense investigations for several years. Here we report the development of a whole-cell medium throughput screening technology for the discovery of CYP11B2 inhibitors. The new screening system displayed high reproducibility and was applied to investigate a library of pharmacologically active compounds. 1268 compounds were investigated during this study which revealed 5 selective inhibitors of CYP11B2 (after validation against CYP11B1). The new inhibitors of CYP11B2 are already existing drugs that could be used either in the treatment of hyperaldosteronism-related diseases or as lead compounds that could further be optimised to achieve safer and selective inhibitors of aldosterone synthase. Article from the Special issue on 'Targeted Inhibitors'.

Coexpression of CYP11B2 or CYP11B1 with adrenodoxin and adrenodoxin reductase for assessing the potency and selectivity of aldosterone synthase inhibitors.

Excessive production of aldosterone has been implicated in the pathogenesis of hypertension and heart failure. One approach to ameliorate the deleterious effects of aldosterone is to suppress its biosynthesis. The enzyme aldosterone synthase (CYP11B2) is responsible for the final step of aldosterone synthesis. It requires electron transfer from the adrenodoxin/adrenodoxin reductase system to catalyze the production of aldosterone. A stable cell line simultaneously overexpressing recombinant human CYP11B2 as well as human adrenodoxin and adrenodoxin reductase was established to help maximize the enzyme activity. The homogenate of these cells was used to develop an in vitro CYP11B2 assay using 11-deoxycorticosterone as a substrate. By the same strategy, another stable cell line simultaneously overexpressing human 11beta-hydroxylase (CYP11B1), an enzyme responsible for the final step of cortisol biosynthesis, and the two electron transfer proteins was also established, and an in vitro CYP11B1 assay using 11-deoxycortisol as a substrate was likewise developed to assess the selectivity of CYP11B2 inhibitors. FAD286, a reference CYP11B2 inhibitor, inhibited CYP11B2 and CYP11B1 activities with IC(50) values of 1.6+/-0.1 and 9.9+/-0.9 nM (mean+/-SEM, n=3-6), respectively. Kinetics studies revealed that the compound inhibited the activity of both enzymes competitively with respective K(i) values of 0.8+/-0.04 and 2.2+/-0.2 nM (n=3-4). These assays can be used for assessing the potency and selectivity of CYP11B2 inhibitors for the treatment of hypertension and heart failure.

Torcetrapib induces aldosterone and cortisol production by an intracellular calcium-mediated mechanism independently of cholesteryl ester transfer protein inhibition.

ILLUMINATE (Investigation of Lipid Level Management to Understand its Impact in Atherosclerotic Events), the phase 3 morbidity and mortality trial of torcetrapib, a cholesteryl ester transfer protein (CETP) inhibitor, identified previously undescribed changes in plasma levels of potassium, sodium, bicarbonate, and aldosterone. A key question after this trial is whether the failure of torcetrapib was a result of CETP inhibition or of some other pharmacology of the molecule. The direct effects of torcetrapib and related molecules on adrenal steroid production were assessed in cell culture using the H295R as well as the newly developed HAC15 human adrenal carcinoma cell lines. Torcetrapib induced the synthesis of both aldosterone and cortisol in these two in vitro cell systems. Analysis of steroidogenic gene expression indicated that torcetrapib significantly induced the expression of CYP11B2 and CYP11B1, two enzymes in the last step of aldosterone and cortisol biosynthesis pathway, respectively. Transcription profiling indicated that torcetrapib and angiotensin II share overlapping pathways in regulating adrenal steroid biosynthesis. Hormone-induced steroid production is mainly mediated by two messengers, calcium and cAMP. An increase of intracellular calcium was observed after torcetrapib treatment, whereas cAMP was unchanged. Consistent with intracellular calcium being the key mediator of torcetrapib's effect in adrenal cells, calcium channel blockers completely blocked torcetrapib-induced corticoid release and calcium increase. A series of compounds structurally related to torcetrapib as well as structurally distinct compounds were profiled. The results indicate that the pressor and adrenal effects observed with torcetrapib and related molecules are independent of CETP inhibition.

[Semen descurainiae inhibits CYP11B1, CYP11B2 and TGF-beta1 mRNA expression in left ventricular].

OBJECTIVE: To investigate the effects of Semen descurainiae and Captopril on CYP11B1, CYP11B2 and TGF-beta1 mRNA expression of heart tissue in rats treated with Abdominal Aortic Banding. METHODS: Ventricular remodeling was induced by abdominal aortic banding (AAB) in rats. After 30 days' treatment, the ratios of LVW/BW (left ventricle weight/body weight), HW/BW (heart weight/body weight) were calculated; Then the CYP11B, CYP11B2 and TGF-beta1 mRNA expression of left ventricle were detected by Real-time PCR, respectively. RESULTS: The experimental data demonstrated that Semen descurainiae decreased the indexes of LVW/BW and HW/BW, down-regulated CYP11B, CYP11B2 and TGF-beta1 mRNA expression in left ventricle (P<0.05). CONCLUSION: Semen desceurainiae can significantly inhibit the experimental ventricular remodeling; the mechanism is related to its ability to attenuate the mRNA expression of CYP11B1, CYP11B2 and TGF-beta1 in left ventricle. The inhibition of aldosterone key gene expression by Semen descurainiae may contribute to its effect on restraint cardiac remodeling.

Aldosterone synthase (CYP11B2)-344T/C polymorphism and renoprotective response to losartan treatment in diabetic nephropathy.

OBJECTIVE: It has been suggested that an aldosterone synthase gene polymorphism (CYP11B2 -344T/C) is predictive of the blood pressure lowering effect of angiotensin II receptor blockers in essential hypertension. We investigated whether this polymorphism is predictive of reductions in blood pressure and albuminuria and preservation of glomerular filtration rate (GFR) during short-term and long-term treatment with losartan in 57 hypertensive type-1 diabetic patients with diabetic nephropathy. MATERIAL AND METHODS: After a 4-week washout period, patients received losartan (100 mg o.d.) and were followed for a mean follow-up of 36 months. At baseline, after 2 and 4 months, and every 6 months thereafter, GFR (51Cr-EDTA-clearance), albuminuria and 24-h blood pressure were determined. The CYP11B2 -344T/C polymorphism was determined by standard polymerase chain reaction (PCR). RESULTS: The TT, CT and CC genotypes were found in 28 %, 58 % and 14 % of patients, respectively. At baseline albuminuria and blood pressure did not differ between genotype groups. Plasma aldosterone levels (geometric mean (95 % CI)) were similar at baseline: 87 (60-125), 77 (53-112), and 89 (49-161) pg mL(-1) and during follow-up (not significant). After initiation of losartan treatment, comparable mean (SE) reductions in blood pressure and albuminuria were seen in patients with TT, CT and CC genotypes (p >0.6 between groups). After long-term follow-up, there was a tendency towards a difference in systolic blood pressure reduction (p = 0.07, one-way ANOVA), suggesting a poorer response in patients with the CC genotype. No significant difference in rate of decline in GFR (median (range)) was seen between groups (TT, CT, CC): 4.2 (-1.0 to 16.0), 3.2 (-1.6 to 13.8) and 2.6 (-0.1 to 11.0) mL min(-1)year(-1), respectively (p = 0.5). CONCLUSIONS: Compared to a previous smaller study of angiotensin II receptor blockade in essential hypertension, we could not confirm that CYP11B2 -344T/C genotypes contribute towards explaining the observed variability in response to treatment with angiotensin II receptor blockers, which could be due to lack of power.

Development of test systems for the discovery of selective human aldosterone synthase (CYP11B2) and 11beta-hydroxylase (CYP11B1) inhibitors. Discovery of a new lead compound for the therapy of congestive heart failure, myocardial fibrosis and hypertension.

Two key players in adrenal steroid hormone biosynthesis are the human mitochondrial cytochrome P450 enzymes CYP11B1 and CYP11B2 that catalyze the final steps in the biosynthesis of cortisol and aldosterone, respectively. Overproduction of both hormones contributes to a number of severe diseases, as illustrated by the association of elevated aldosterone levels with hypertension and higher mortality in congestive heart failure, and by Cushing's syndrome as the clinical correlate of chronic hypercortisolism. Thus, CYP11B1 and CYP11B2 comprise new targets for drug treatment and selective inhibitors of both enzymes are of high pharmacological interest. To facilitate the search for such compounds, we have established novel test procedures using recombinant fission yeast strains that stably express these enzymes. The aim of this study was to compare the inhibition profiles displayed by these enzymes in established mammalian cell culture test systems to those obtained with the new fission yeast assays, and to evaluate the usefulness of the Schizosaccharomyces pombe strains as screening systems for the identification of novel lead compounds. Using these test systems, we were able to identify a new and very selective CYP11B2 inhibitor (SIAS-1) that displayed no detectable interference with CYP11B1 activity.

Development of a test system for inhibitors of human aldosterone synthase (CYP11B2): screening in fission yeast and evaluation of selectivity in V79 cells.

Aldosterone synthase (CYP11B2) is a mitochondrial cytochrome P450 enzyme catalyzing the last steps of aldosterone production in the adrenal cortex. A new pharmacological approach for the treatment of the aldosterone induced effects in congestive heart failure and all forms of hyperaldosteronism could be the use of CYP11B2 inhibitors. In search for such compounds, it was our goal to develop a cellular enzyme assay suitable for screening high numbers of compounds. An assay procedure for the evaluation of inhibitors using the human CYP11B2 expressed in fission yeast Schizosaccharomyces pombe was established and a series of 10 compounds was tested in this whole cellular system. Human 11beta-hydroxylase (CYP11B1), which catalyzes the production of glucocorticoids, shows more than 90% homology compared to human CYP11B2. As this enzyme should not be affected, strong inhibitors of CYP11B2 have to be tested for selectivity. For that purpose, an assay procedure with V79MZ cells that express human CYP11B1 and CYP11B2, respectively, was integrated into the evaluation process. Using these screening procedures a potent and rather selective non-steroidal inhibitor of human CYP11B2 was detected with an IC(50) value of 59nM. We also identified a very potent inhibitor of both enzymes showing a stronger inhibitory activity against the cortisol producing CYP11B1.

Deletion hybrid genes, due to unequal crossing over between CYP11B1 (11beta-hydroxylase) and CYP11B2(aldosterone synthase) cause steroid 11beta-hydroxylase deficiency and congenital adrenal hyperplasia.

Chromosomal rearrangements are natural experiments that can provide unique insights into in vivo regulation of genes and physiological systems. We have studied a patient with congenital adrenal hyperplasia and steroid 11beta-hydroxylase deficiency who was homozygous for a deletion of the CYP11B1 and CYP11B2 genes normally required for cortisol and aldosterone synthesis, respectively. The genes were deleted by unequal recombination between the tandemly arranged CYP11B genes during a previous meiosis, leaving a single hybrid gene consisting of the promoter and exons 1-6 of CYP11B2 and exons 7-9 of CYP11B1. The hybrid gene also carried an I339T mutation formed by intracodon recombination at the chromosomal breakpoint. The mutant complementary DNA corresponding to this gene was expressed in COS-1 cells and was found to have relatively unimpaired 11beta-hydroxylase and aldosterone synthase activities. Apparently the 11beta-hydroxylase deficiency and the adrenal hyperplasia are due to the lack of expression of this gene in the adrenal zona fasciculata/reticularis resulting from replacement of the CYP11B1 promoter and regulatory sequences by those of CYP11B2.

[CYP11B2 expression in rat liver and the efficacy of antisterone on liver fibrosis].

OBJECTIVE: To identify aldosterone synthase gene-CYP11B2 mRNA expression in rat livers and evaluate the efficacy of antisterone. METHODS: One hundred and twenty male Wistar rats were divided into 3 groups: model group (subcutaneous injection of CCl(4), 0.25 ml/100 mg, 3 times/week), antisterone group (besides same dosage of CCl(4) given, gastropufusion of antisterone of 20mg x kg(-1) x d(-1)), and control group (subcutaneous injection of olive oil). The area of collagen was examined by image analyzing system. The expression of aldosterone synthase gene-CYP11B2 mRNA was detected by RT-PCR and in situ hybridization. RESULTS: The expression of CYP11B2 mRNA, which located in the endoplasm of HSC, was upregulated when fibrogenesis occurred. The grade of fibrosis and the area of collagen in antisterone group were less than those in model group before 6th week (P<0.05). After the 6th week however, there was no significant difference between the two groups (P>0.05). CONCLUSIONS: The expression of CYP11B2 mRNA is upregulated in fibrotic liver. Antisterone can partly have a fibrogenesis-inhibiting effect on the early stage of CCl(4)-induced hepatic fibrosis.

Type 1 aldosterone synthase deficiency presenting in a middle-aged man.

Aldosterone synthase deficiency due to mutations in the CYP11B2 gene usually presents in infancy with electrolyte abnormalities and failure to thrive, whereas affected adults are usually asymptomatic. We describe a patient who first came to medical attention in middle age when he developed hyperkalemia after preparation for a barium enema. Past medical history was notable for failure to thrive in infancy. He had elevated PRA with low serum and urinary levels of aldosterone and its metabolites and normal or slightly elevated levels of 18-hydroxycorticosterone. These findings suggested a diagnosis of type 1 aldosterone synthase deficiency. The patient had a homozygous duplication of six nucleotides at codon 143 in exon 3 of CYP11B2, leading to the insertion of two amino acid residues (Arg-Leu). When the corresponding mutant complementary DNA was expressed in cultured cells, the resulting enzyme was completely inactive, confirming the diagnosis. We conclude that aldosterone synthase deficiency represents an unusual cause of hyperreninemic hypoaldosteronism presenting in adult life, but it should be suspected if the past medical history is positive for failure to thrive in childhood or if the patient manifests no other recognized causes of hyperreninemic hypoaldosteronism.

Autocrine-paracrine role of endothelin-1 in the regulation of aldosterone synthase expression and intracellular Ca2+ in human adrenocortical carcinoma NCI-H295 cells.

The role played by endothelin (ET-1) and its receptor subtypes A and B (ET(A) and ET(B)) in the functional regulation of human NCI-H295 adrenocortical carcinoma cells has been investigated. Reverse transcription-PCR with primers specific for prepro-ET-1, human ET-1 converting enzyme-1, ET(A), and ET(B) complementary DNAs consistently demonstrated the expression of all genes in NCI-H295 cells. The presence of mature ET-1 and both its receptor subtypes was confirmed by immunocytochemistry and autoradiography, respectively. Aldosterone synthase (AS) messenger RNA was also detected in NCI-H295 cells, and AS gene expression was enhanced by both ET-1 and the specific ET(B) agonist IRL-1620; this effect was not inhibited by either the ET(A) antagonist BQ-123 or the ET(B) antagonist BQ-788. A clear-cut increase in the intracellular Ca2+ concentration in NCI-H295 cells in response to ET(B), but not ET(A), activation was observed. In light of these findings, the following conclusions can be drawn: 1) NCI-H295 cells possess an active ET-1 biosynthetic pathway and are provided with ET(A) and ET(B) receptors; 2) ET-1 regulates in an autocrine/paracrine fashion the secretion of aldosterone by NCI-H295 cells by enhancing both AS transcription and raising the intracellular Ca2+ concentration; and 3) the former effect of ET-1 probably involves the activation of both receptor subtypes, whereas calcium response is exclusively mediated by the ET(B) receptor.

In vivo regulation of gene expression of enzymes controlling aldosterone synthesis in rat adrenal.

We studied the effect of alterations in the intake of sodium and potassium as well as changes in circulating adrenocorticotropin (ACTH) on the expression of the two rate-limiting systems of aldosterone formation in the rat. Low sodium and high potassium intake promoted time-dependent increases in the zona glomerulosa cytochrome P450scc (P450scc) and cytochrome P450c11 (P450c11) protein and mRNA levels, but no changes were found in the zona fasciculata-reticularis. In addition, these responses were associated with markedly elevated transcriptional activities. To further define the contribution of P450c11 and P450c18 (aldosterone synthase) in response to these differing intakes, we evaluated their mRNA levels using gene-specific oligonucleotide probes. P450c18 mRNA was restricted to the zona glomerulosa, whereas P450c11 mRNA was detected in both zona glomerulosa and zona fasciculata-reticularis. Furthermore, only P450c18 mRNA was induced by both low sodium or high potassium intake, as P450c11 mRNA levels remained unchanged. Captopril, an inhibitor of angiotensin-I converting enzyme, abolished the enhancing effects of the low sodium regimen on P450scc and P450c18 mRNA levels. Captopril also suppressed the augmentation of P450c18 mRNA observed with potassium supplementation but had no effect on P450scc mRNA levels. When the hypocholesterolemic drug 4-aminopyrazolopyrimidine (4-APP) was administered to rats for 3 consecurive days, both the level of plasma ACTH and the adrenal content of mRNA encoding P450scc increased 24 h post final injection. The coadministration of dexamethasone with 4-APP prevented these increases. In contrast, the mRNA content of P450c11 remained at control levels. In conclusion, this work demonstrates that variations in the intake of sodium and potassium act on the expression of the CYP11B2 gene, but not on that of the CYP11B1 gene. Moreover angiotensin-II (A-II) is an important factor in this mechanism of action. Both ions also enhance the expression of the CYP11A1 gene. A-II appears to participate in the mechanism of action of the low sodium intake at this level. Another mechanism is postulated for the action of potassium supplementation since captopril did not prevent the increased expression of the CYP11A1 gene. In addition, the fact that 4-APP enhanced the mRNA level of P450scc but not that of P450c, also demonstrates different regulation of the P450s involved at the early and final steps of aldosteroone formation in the rat adrenal zona glomerulosa in vivo.