RESUMO
Angiotensin II (AngII) stimulates aldosterone biosynthesis in the zona glomerulosa of the adrenal cortex. AngII also triggers the MAPK pathways (ERK1/2 and p38). Because ERK1/2 phosphorylation is a transient process, phosphatases could play a crucial role in the acute steroidogenic response. Here we show that the dual specificity (threonine/tyrosine) MAPK phosphatase-1 (MKP-1) is present in bovine adrenal glomerulosa cells in primary culture and that AngII markedly increases its expression in a time- and concentration-dependent manner (IC(50) = 1 nm), a maximum of 548 +/- 10% of controls being reached with 10 nm AngII after 3 h (n = 3, P < 0.01). This effect is completely abolished by losartan, a blocker of the AT(1) receptor subtype. Moreover, this AngII-induced MKP-1 expression is reduced to 250 +/- 35% of controls (n = 3, P < 0.01) in the presence of U0126, an inhibitor of ERK1/2 phosphorylation, suggesting an involvement of the ERK1/2 MAPK pathway in MKP-1 induction. Indeed, shortly after AngII-induced phosphorylation of ERK1/2 (220% of controls at 30 min), MKP-1 protein expression starts to increase. This increase is associated with a reduction in ERK1/2 phosphorylation, which returns to control values after 3 h of AngII challenge. Enhanced MKP-1 expression is essentially due to a stabilization of MKP-1 mRNA. AngII treatment leads to a 53-fold increase in phosphorylated MKP-1 levels and a doubling of MKP-1 phosphatase activity. Overexpression of MKP-1 results in decreased phosphorylation of ERK1/2 and aldosterone production in response to AngII stimulation. These results strongly suggest that MKP-1 is the specific phosphatase induced by AngII and involved in the negative feedback mechanism ensuring adequate ERK1/2-mediated aldosterone production in response to the hormone.
Assuntos
Angiotensina II/farmacologia , Fosfatase 1 de Especificidade Dupla/genética , Mineralocorticoides/biossíntese , Zona Glomerulosa/efeitos dos fármacos , Animais , Bovinos , Células Cultivadas , Relação Dose-Resposta a Droga , Retroalimentação Fisiológica/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação , Estabilidade de RNA/efeitos dos fármacos , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Fatores de Tempo , Transfecção , Zona Glomerulosa/metabolismoRESUMO
We have identified a novel cytosine/thymidine polymorphism of the human steroidogenic acute regulatory (StAR) gene promoter located 3 bp downstream of the steroidogenic factor-1 (SF-1)-binding site and 9 bp upstream of the TATA box (ATTTAAG). Carriers of this mutation have a high prevalence of primary aldosteronism. In transfection experiments, basal StAR promoter activity was unaltered by the mutation in murine Y-1 cells and human H295R cells. In Y-1 cells, forskolin (25 microM, 6 h) significantly increased wild-type promoter activity to 230+/-33% (P<0.05, n=4). In contrast, forskolin increased mutated promoter activity only to 150+/-27%, with a significant 35% reduction compared to wild type (P<0.05, n=3). In H295R cells, angiotensin II (AngII; 10 nM) increased wild-type StAR promoter activity to 265+/-22% (P<0.01, n=3), while mutated StAR promoter activity in response to AngII only reached 180+/-29% of controls (P< 0.01, n=3). Gel mobility shift assays show the formation of two additional complexes with the mutated promoter: one with the transcription repressor DAX-1 and another with a yet unidentified factor, which strongly binds the SF-1 response element. Thus, this novel mutation in the human StAR promoter is critically involved in the regulation of StAR gene expression and is associated with reduced promoter activity, a finding relevant for adrenal steroid response to physiological stimulators.
Assuntos
Mutação , Fosfoproteínas/genética , Polimorfismo Genético , Regiões Promotoras Genéticas , Animais , Sequência de Bases , Fatores de Transcrição COUP/metabolismo , Linhagem Celular , AMP Cíclico/metabolismo , Citosina/metabolismo , Proteínas de Homeodomínio/metabolismo , Humanos , Camundongos , Dados de Sequência Molecular , Fosfoproteínas/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Alinhamento de Sequência , Fator de Transcrição Sp1/metabolismo , Fator Esteroidogênico 1 , Timidina/metabolismo , Fatores de Transcrição/metabolismoRESUMO
The octapeptide hormone, angiotensin II (AngII) and ACTH stimulate mineralocorticoid biosynthesis in the zona glomerulosa of the adrenal cortex in part by promoting the transcription of the gene coding for the steroidogenic acute regulatory (StAR) protein. We have examined whether chicken ovalbumin upstream promoter-transcription factor (COUP-TF), a member of the orphan nuclear receptor family of transcription factors, is involved in this transcriptional regulation. We analyzed COUP-TF and StAR mRNA and protein levels in bovine adrenal glomerulosa cells in primary culture. COUP-TF protein was readily detectable in nonstimulated cells, and AngII markedly reduced its expression in a time- and concentration-dependent manner (IC50 = 1 nm), to 46 +/- 4.4% of control levels after 6 h, (n = 3; P < 0.01). This repression was paralleled by a marked decrease in COUP-TF mRNA levels, reaching 18 +/- 8.8% of controls (n = 3, P < 0.01) after 6 h and by a 20-fold increase in aldosterone output. In bovine glomerulosa cells overexpressing COUP-TFI and -II, the induction of StAR mRNA and protein elicited by AngII was completely suppressed to control levels, and the aldosterone response was significantly reduced (from 4.8 +/- 1.1-fold the basal value in mock-infected cells to 1.9 +/- 0.5-fold and 2.2 +/- 0.7-fold in COUP-TFI- and COUP-TFII-expressing cells, respectively; n = 3; P < 0.01 for both differences). Finally, by using electrophoretic mobility shift assays and chromatin immunoprecipitation, we have shown a direct interaction between COUP-TF and the proximal StAR promoter. These results suggest that COUP-TF exerts a tonic inhibition on steroidogenesis by repressing StAR protein expression and that activators of aldosterone biosynthesis lift this inhibition in part by repressing COUP-TF levels.
Assuntos
Aldosterona/biossíntese , Proteínas de Ligação a DNA/metabolismo , Receptores de Esteroides/metabolismo , Fatores de Transcrição/metabolismo , Zona Glomerulosa/metabolismo , Aldosterona/metabolismo , Angiotensina II/farmacologia , Animais , Fator I de Transcrição COUP , Fator II de Transcrição COUP , Fatores de Transcrição COUP , Bovinos , Células Cultivadas , Galinhas , Colforsina/farmacologia , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica , Mineralocorticoides/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Regiões Promotoras Genéticas/genética , Estabilidade de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Esteroides/genética , Fatores de Transcrição/genética , Zona Glomerulosa/efeitos dos fármacosRESUMO
In addition to intracellular cholesterol synthesis, plasma low- and high-density lipoproteins (LDL and HDL, respectively) are the major potential sources of a cholesterol precursor for steroid synthesis in all steroidogenic tissues. LDL- and HDL-cholesterol are taken up by cells through entirely distinct mechanisms. In the case of aldosterone production in the zona glomerulosa of the adrenal cortex, it has been assumed in the past that LDL is the major supplier of cholesterol. However, recent developments, in particular the discovery of the scavenger receptor class B type I for HDL and the characterization of its properties, have questioned this view. In fact, the nature of the challenging factor (angiotensin II or adrenocorticotropic hormone) appears to determine which pool of cholesterol is preferentially mobilized and which pathway (LDL receptor endocytosis or selective uptake through the HDL receptor) is regulated.
Assuntos
Aldosterona/biossíntese , Ésteres do Colesterol/metabolismo , LDL-Colesterol/metabolismo , Colesterol/metabolismo , Lipoproteínas HDL/metabolismo , Proteínas de Membrana , Receptores Imunológicos , Receptores de Lipoproteínas , Hormônio Adrenocorticotrópico/farmacologia , Angiotensina II/farmacologia , Animais , Antígenos CD36/metabolismo , Colesterol/biossíntese , Humanos , Mineralocorticoides/metabolismo , Receptores Depuradores , Receptores Depuradores Classe B , Zona Glomerulosa/citologia , Zona Glomerulosa/efeitos dos fármacos , Zona Glomerulosa/metabolismoRESUMO
We examined whether the mRNA for steroidogenic acute regulatory (StAR) protein, a crucial factor in the rate-limiting step of aldosterone biosynthesis, is expressed and regulated in rat heart. We performed quantitative RT-PCR for StAR mRNA in an in vitro and an in vivo model: purified rat neonatal cardiomyocytes in primary culture and myocardial infarction (MI) in the rat. StAR mRNA was expressed in cultured cardiomyocytes, and angiotensin II (10 nM) increased it in a time-dependent fashion (132 +/- 2.7% of controls after 24 h; n = 3; P < 0.05). Concomitantly, angiotensin II stimulated aldosterone production in the culture medium from 32.6 +/- 6.1 to 54 +/- 12.7 fmol/mg protein after 24 h (n = 8; P < 0.05). StAR mRNA levels in cardiomyocytes were dramatically reduced after 24-h treatment with dexamethasone in a concentration-dependent manner (50% inhibitory concentration, 10 nM); maximal inhibition (to 15 +/- 6% of control; P < 0.001; n = 6) was achieved with 100 nM dexamethasone. This inhibition was prevented by RU486. In the rat MI model, StAR mRNA was also present in control heart tissue and was increased 2.4-fold in the noninfarcted area of the left ventricle after MI (n = 6; P < 0.01). This effect was completely prevented by treatment with losartan (8 mg/kg per d) and spironolactone (80 mg/kg per d), which reduced StAR mRNA levels to values not different from those in non-MI controls. Thus, the mRNA for an indispensable factor in aldosterone biosynthesis, the StAR protein, is expressed in the rat heart and is up-regulated after MI. These results support the view of a local synthesis of aldosterone in the heart and of intracrine/paracrine deleterious effects of the mineralocorticoid in heart failure.
Assuntos
Infarto do Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Fosfoproteínas/metabolismo , Aldosterona/farmacologia , Angiotensina II/farmacologia , Animais , Células Cultivadas , Dexametasona/farmacologia , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/fisiologia , Glucocorticoides/farmacologia , Masculino , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosfoproteínas/genética , RNA Mensageiro/metabolismo , Ratos , Ratos WistarRESUMO
In adrenal glomerulosa cells, the stimulation of aldosterone biosynthesis by angiotensin II (Ang II) occurs via activation of the Ca2+ messenger system, increased expression of the steroidogenic acute regulatory protein, and enhanced transfer of cholesterol to the inner mitochondrial membrane. We examined here whether Ang II affects the activity of cholesterol ester hydrolase (CEH), also named hormone-sensitive lipase, the enzyme recruiting cholesterol from intracellular pools, in bovine adrenal glomerulosa cells. In bovine adrenal tissue, CEH activity was detected with characteristics similar to those reported in other tissues (Michaelis constant = 46.3 +/- 6.7 microM, n = 3; maximal velocity = 1 nmol/mg.min). This activity was significantly enhanced in isolated bovine glomerulosa cells challenged for 2 h with 10 nM Ang II (to 149 +/- 11% of controls, n = 3). Similarly, 25 microM forskolin raised CEH activity to 151 +/- 5% of controls (n = 3). This increase in activity of CEH was not due to an increase in the amount of enzyme protein but was associated with an increased phosphorylation of the enzyme to 337 +/- 33% of controls (n = 9, P < 0.0001). Potassium ion (K+) and forskolin also stimulated [32P]orthophosphate incorporation, although to a lesser extent (to 157 +/- 18% and 186 +/- 25% of controls, respectively). On SDS-PAGE, the majority of this radioactivity was associated with a species of 172 kDa, corresponding to a CEH dimer. Both Ang II-induced CEH phosphorylation and pregnenolone production were significantly reduced (to 47 +/- 6% and 50 +/- 8% of controls with Ang II alone, respectively) in the presence of PD098059, an inhibitor of p42/p44 MAPK. Indeed, Ang II challenge led to a rapid 32P incorporation into p42/p44 MAPK. These results demonstrate that, in addition to its known effects on intramitochondrial cholesterol transfer, Ang II also promotes aldosterone biosynthesis by rapidly increasing cholesterol supply to the outer mitochondrial membrane.
Assuntos
Angiotensina II/farmacologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Esterol Esterase/metabolismo , Zona Glomerulosa/enzimologia , Animais , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Bovinos , Células Cultivadas , Colforsina/farmacologia , Ativação Enzimática/efeitos dos fármacos , Proteína Quinase 3 Ativada por Mitógeno , Fosforilação/efeitos dos fármacos , Potássio/farmacologia , Proteína Quinase C/metabolismo , Zona Glomerulosa/citologiaRESUMO
In the adrenal glomerulosa cell, aldosterone is synthesized from cholesterol, which is supplied to the cell and stored under the form of cholesterol esters, then hydrolyzed to be transferred to the mitochondrial outer membrane and finally transported to the inner membrane where the P450 side-chain cleavage enzyme will convert it to pregnenolone. Angiotensin II (AngII), one of the major physiological regulators of mineralocorticoid synthesis, appears to affect most of the steps along this cascade and thus to exert a powerful control over the use of cholesterol for aldosterone production.
Assuntos
Aldosterona/biossíntese , Angiotensina II/fisiologia , Ésteres do Colesterol/metabolismo , Enzima de Clivagem da Cadeia Lateral do Colesterol/metabolismo , Mitocôndrias/enzimologia , Zona Glomerulosa/metabolismo , Animais , Transporte Biológico Ativo/fisiologia , Bovinos , Humanos , Camundongos , Mineralocorticoides/biossíntese , Pregnenolona/biossíntese , RatosRESUMO
The oscillatory nature of the intracellular calcium signal has been recognized as soon as the methodological developments allowed us to record calcium fluctuations at the single cell level. While the principal mechanisms responsible for the generation of these oscillations have been partially resolved, more attention has been recently focused on signal decoding and more particularly on the role of cell structure organization in transducing this signal to the molecular targets of the calcium messenger.
Assuntos
Sinalização do Cálcio/fisiologia , Animais , Citosol/fisiologia , Desenho de Fármacos , Retroalimentação Fisiológica , Humanos , Mitocôndrias/fisiologia , Transdução de Sinais/fisiologiaRESUMO
OBJECTIVE: The increased mortality observed with the cholesteryl ester transfer protein inhibitor torcetrapib is partly due to increased aldosterone production and blood pressure. The mechanisms underlying these effects were investigated. METHODS: Cytochrome P450 subunit 11B2 (aldosterone synthase), extracellular signal-regulated kinase (p44/42) and voltage-gated Cachannel alpha subunit mRNA profiling, aldosterone production, cytosolic calcium and RNA interference were assessed in adrenocarcinoma human cells (H295R). Telemetry was conducted in spontaneously hypertensive rats. RESULTS: Torcetrapib and angiotensin II (Ang II) but not dalcetrapib (a structurally different cholesteryl ester transfer protein inhibitor) elevated both cytochrome P450 subunit 11B2 mRNA and aldosterone production in H295R cells at 6 h. At days 1-5, torcetrapib produced a sustained increase of cytochrome P450 subunit 11B2 mRNA, unlike Ang II. Although torcetrapib and Ang II potentiated the effect of 25-OH cholesterol and raised pregnenolone levels, torcetrapib increased neither cytosolic Ca at 5 min nor extracellular signal-regulated kinase1/2 phosphorylation, suggesting initially divergent pathways. Unlike Ang II, torcetrapib steroidogenesis was not affected by Ang II type 1 receptor antagonism or voltage-gated T-type Ca channel antagonism, but was blocked by several L-type Cachannel antagonists. In unbiased genome-wide screening, Ang II and torcetrapib modulated an overlapping but distinct set of genes in H295R cells. Torcetrapib, but not Ang II, upregulated mRNA levels of the L-type Ca channel alpha 1C subunit. In spontaneously hypertensive rat, torcetrapib had a potent hypertensive effect mediated by the L-type Ca channel. CONCLUSION: The unique steroidogenic and hypertensive side effects of torcetrapib may be linked and involve voltage-gated L-type Ca channels. Structurally unrelated cholesteryl ester transfer protein inhibitors such as dalcetrapib do not share this effect.
Assuntos
Anticolesterolemiantes/farmacologia , Canais de Cálcio Tipo L/efeitos dos fármacos , Proteínas de Transferência de Ésteres de Colesterol/antagonistas & inibidores , Hipertensão/tratamento farmacológico , Quinolinas/farmacologia , Córtex Suprarrenal/efeitos dos fármacos , Córtex Suprarrenal/metabolismo , Córtex Suprarrenal/patologia , Neoplasias das Glândulas Suprarrenais , Aldosterona/metabolismo , Amidas , Angiotensina II/farmacologia , Animais , Pressão Sanguínea/efeitos dos fármacos , Pressão Sanguínea/fisiologia , Cálcio/metabolismo , Canais de Cálcio Tipo L/metabolismo , Linhagem Celular Tumoral , Citocromo P-450 CYP11B2/biossíntese , Citocromo P-450 CYP11B2/genética , Citosol/efeitos dos fármacos , Citosol/metabolismo , Indução Enzimática/efeitos dos fármacos , Ésteres , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Expressão Gênica/efeitos dos fármacos , Perfilação da Expressão Gênica , Humanos , Canal de Sódio Disparado por Voltagem NAV1.1 , Proteínas do Tecido Nervoso/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Endogâmicos SHR , Canais de Sódio/metabolismo , Relação Estrutura-Atividade , Compostos de Sulfidrila/farmacologiaRESUMO
Angiotensin II (AngII), potassium ion, and ACTH are the main factors controlling aldosterone biosynthesis in adrenal glomerulosa cells. AP-1 response elements for the immediate early gene products, c-Fos and c-Jun, have been identified, among others, in the promoter of the steroidogenic acute regulatory (StAR) protein gene, whose expression is acutely regulated by activators of aldosterone production. In bovine glomerulosa cells, AngII treatment led to a rapid and transient increase in c-fos mRNA expression, c-Fos protein expression, and c-Fos phosphorylation. Inhibition of the ERK1/2 MAPK pathway abolished the effect of AngII on c-fos mRNA, protein, and phosphorylation. EMSA and chromatin immunoprecipitation experiments demonstrated that c-Fos binds with c-Jun to the proximal StAR promoter and that AngII treatment increases the amount of c-Fos bound to the promoter. Overexpression of a dominant-negative form of c-Fos with adenoviral vectors inhibited StAR mRNA and StAR protein expression as well as aldosterone biosynthesis in response to AngII. The dominant-negative c-Fos also prevented the increase in protein synthesis induced by AngII in glomerulosa cells, as assessed by [(3)H]leucine incorporation. These results indicate that AngII rapidly induces c-Fos expression and posttranslational modifications. Furthermore, a heterodimeric c-Fos/c-Jun complex binds to the proximal StAR promoter in glomerulosa cells, thus activating StAR gene expression and acute aldosterone biosynthesis. Finally, c-Fos also contributes to other functional responses to the hormone, such as protein synthesis.
Assuntos
Aldosterona/metabolismo , Angiotensina II/farmacologia , Genes fos/fisiologia , Biossíntese de Proteínas , Zona Glomerulosa/metabolismo , Animais , Bovinos , Células Cultivadas , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Genes fos/efeitos dos fármacos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilação , Regiões Promotoras Genéticas , Ligação Proteica , Biossíntese de Proteínas/efeitos dos fármacos , Biossíntese de Proteínas/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Zona Glomerulosa/efeitos dos fármacosRESUMO
Efficacy and safety data for dalcetrapib (RO4607381/JTT-705) are presented, following a report of increased mortality and cardiac events with another cholesteryl ester transfer protein inhibitor, torcetrapib, associated with off-target adverse effects (hypertension and the activation of the renin-angiotensin-aldosterone system). The efficacy and clinical safety of dalcetrapib 300, 600, and 900 mg or placebo were assessed (n = 838) in 4 pooled 4-week phase IIa trials (1 monotherapy, n = 193; 3 statin combination, n = 353) and 1 12-week phase IIb trial (with pravastatin, n = 292). Nonclinical safety, assessed by the induction of aldosterone production and aldosterone synthase (cytochrome P450 11B2) messenger ribonucleic acid, was measured in human adrenocarcinoma (H295R) cells exposed to dalcetrapib or torcetrapib. Dalcetrapib increased high-density lipoprotein cholesterol by up to 36% and apolipoprotein A-I by up to 16%. The incidence of adverse events (AEs) was similar between placebo (42%) and dalcetrapib 300 mg (50%) and 600 mg (42%), with more events with dalcetrapib 900 mg (58%) (p <0.05, pooled 4-week studies). Six serious AEs (3 with placebo, 1 with dalcetrapib 300 mg, and 2 with dalcetrapib 600 mg) were considered "unrelated" to treatment. Cardiovascular AEs were similar across treatment groups, with no dose-related trends and no clinically relevant changes in blood pressure or electrocardiographic results. Findings were similar in the 12-week study. In vitro, torcetrapib but not dalcetrapib increased aldosterone production and cytochrome P450 11B2 messenger ribonucleic acid levels. In conclusion, dalcetrapib alone or in combination with statins was effective at increasing high-density lipoprotein cholesterol and was well tolerated, without clinically relevant changes in blood pressure or cardiovascular AEs and no effects on aldosterone production as assessed nonclinically.
Assuntos
Anticolesterolemiantes/efeitos adversos , Doença da Artéria Coronariana/tratamento farmacológico , Dislipidemias/tratamento farmacológico , Compostos de Sulfidrila/efeitos adversos , Amidas , Anticolesterolemiantes/uso terapêutico , Proteínas de Transferência de Ésteres de Colesterol/antagonistas & inibidores , Método Duplo-Cego , Ésteres , Feminino , Humanos , Incidência , Masculino , Pessoa de Meia-Idade , Quinolinas/efeitos adversos , Quinolinas/uso terapêutico , Medição de Risco , Fatores de Risco , Compostos de Sulfidrila/uso terapêuticoRESUMO
Angiotensin II (Ang II) and adrenocorticotropic hormone stimulate aldosterone biosynthesis in the zona glomerulosa of the adrenal cortex through induction of the expression of the steroidogenic acute regulatory (StAR) protein, which promotes intramitochondrial cholesterol transfer. To understand the mechanism of this induction of the StAR protein, we have examined the effect of Ang II and forskolin, a mimicker of adrenocorticotropic hormone action, on two transcription factors known to modulate StAR gene expression in opposite ways, DAX-1 and SF-1, in bovine adrenal glomerulosa cells in primary culture. Ang II markedly inhibited DAX-1 protein expression in a time- and concentration-dependent manner (to 38.7 +/- 12.9% of controls at 3 nm after 6 h, p < 0.01), an effect that required de novo protein synthesis and ERK2/1 activation. This effect was associated with a concomitant decrease in DAX-1 mRNA and an increase in mitochondrial StAR protein levels. Similarly, forskolin dramatically repressed DAX-1 protein and mRNA expression (to 19.6 +/- 1.8 and 50.3 +/- 4.7% of controls, respectively, p < 0.01). Neither Ang II nor forskolin affected DAX-1 protein and mRNA stability. The aldosterone response to Ang II was markedly reduced (to 59 +/- 4% of controls, p < 0.01) in transiently transfected cells overexpressing DAX-1. Whereas Ang II was without effect on SF-1 expression, forskolin significantly increased SF-1 protein and mRNA levels in a cycloheximide-sensitive manner (to 167.4 +/- 16.6 and 173.1 +/- 25.1% of controls after 6 h, respectively, p < 0.01). These results demonstrate that the balance between repressor and inducer function of DAX-1 and SF-1 are of critical importance in the regulation of adrenal aldosterone biosynthesis.