Atrial Natriuretic Peptide Inhibits Calcium-Induced Steroidogenic Acute Regulatory Protein Gene Transcription in Adrenal Glomerulosa Cells

Nadia Cherradi, Yves Brandenburger, Michel F. Rossier, Michel B. Vallotton, Douglas M. Stocco and Alessandro M. Capponi

Division of Endocrinology and Diabetology (N.C., Y.B., M.F.R., M.B.V., A.M.C.) Department of Internal Medicine Faculty of Medicine CH-1211 Geneva 14, Switzerland
Department of Cell Biology and Biochemistry (D.M.S.) Texas Tech University Health Sciences Center Lubbock, Texas 79430


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Atrial natriuretic peptide (ANP) is a potent inhibitor of mineralocorticoid synthesis induced in adrenal glomerulosa cells by physiological agonists activating the calcium messenger system, such as angiotensin II (Ang II) and potassium ion (K+). While the role of calcium in mediating Ang II- and K+-induced aldosterone production is clearly established, the mechanisms leading to blockade of this steroidogenic response by ANP remain obscure. We have used bovine adrenal zona glomerulosa cells in primary culture, in which an activation of the calcium messenger system was mimicked by a 2-h exposure to an intracellular high-calcium clamp. The effect of ANP was studied on the following parameters of the steroidogenic pathway: 1) pregnenolone and aldosterone production; 2) changes in cytosolic ([Ca2+]c) and mitochondrial ([Ca2+]m) Ca2+ concentrations, as assessed with targeted recombinant aequorin; 3) cholesterol content in outer mitochondrial membranes (OM), contact sites (CS), and inner membranes (IM); 4) steroidogenic acute regulatory (StAR) protein import into mitochondria by Western blot analysis; 5) StAR protein synthesis, as determined by [35S]methionine incorporation, immunoprecipitation, and SDS-PAGE; 6) StAR mRNA levels by Northern blot analysis with a StAR cDNA; 7) StAR gene transcription by nuclear run-on analysis.

While clamping Ca2+ at 950 nM raised pregnenolone output 3.5-fold and aldosterone output 3-fold, ANP prevented these responses with an IC50 of 1 nM and a maximal effect of 90% inhibition at 10 nM. In contrast, ANP did not affect the [Ca2+]c or [Ca2+]m changes occurring under Ca2+ clamp or Ang II stimulation in glomerulosa cells. The accumulation of cholesterol content in CS (139.7 ± 10.7% of control) observed under high-Ca2+ clamp was prevented by 10 nM ANP (92.4 ± 4% of control). Similarly, while Ca2+ induced a marked accumulation of StAR protein in mitochondria of glomerulosa cells to 218 ± 44% (n = 3) of controls, the presence of ANP led to a blockade of StAR protein mitochondrial import (113.3 ± 15.0%). This effect was due to a complete suppression of the increased [35S]methionine incorporation into StAR protein that occurred under Ca2+ clamp (94.5 ± 12.8% vs. 167.5 ± 17.3%, n = 3). Furthermore, while the high-Ca2+ clamp significantly increased StAR mRNA levels to 188.5 ± 8.4 of controls (n = 4), ANP completely prevented this response. Nuclear run-on analysis showed that increases in intracellular Ca2+ resulted in transcriptional induction of the StAR gene and that ANP inhibited this process.

These results demonstrate that Ca2+ exerts a transcriptional control on StAR protein expression and that ANP appears to elicit its inhibitory effect on aldosterone biosynthesis by acting as a negative physiological regulator of StAR gene expression.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In the zona glomerulosa cells of the adrenal cortex, the octapeptide hormone angiotensin II (Ang II) and potassium (K+) are the most powerful stimuli of aldosterone production. Mineralocorticoid biosynthesis and secretion are highly dependent upon the increase in intracellular calcium triggered by these two agonists through distinct mechanisms (1, 2, 3, 4, 5).

The rate-limiting step in the activation of steroidogenesis is the delivery of cholesterol from the mitochondrial outer membrane to the inner membrane, where the cytochrome P450 side-chain cleavage (P450scc) enzyme is located (6, 7, 8, 9, 10). Earlier studies have shown that cycloheximide, an inhibitor of protein translation, blocked pregnenolone production elicited by either cAMP- or Ca2+-mobilizing hormones (8, 9, 11). Indeed, the appearance of newly synthesized mitochondrial phosphoproteins, referred to as the 30 kDa-proteins, has been shown to correlate directly with steroid production in adrenal cells and MA-10 mouse Leydig tumor cells (12, 13, 14, 15). Currently, the 30-kDa steroidogenic acute regulatory (StAR) protein is considered to be a key regulator of cholesterol delivery to the P450scc enzyme (16, 17, 18, 19). The decisive demonstration came from an inherited disease that leads to a dramatic deficiency in all steroid hormones, congenital lipoid adrenal hyperplasia: mutations in the StAR gene have been shown to underlie this disorder (20, 21).

Atrial natriuretic peptide (ANP), a hormone originally identified in atrial cardiomyocytes, affects blood pressure through its concerted actions on various target organs, including vascular smooth muscle, kidney, and adrenal cortex. In adrenal glomerulosa cells, ANP strongly impairs aldosterone secretion stimulated by Ang II (4, 22, 23, 24, 25), K+ (26), or ACTH (27, 28). In vitro and in vivo studies indicate that ANP-induced inhibition of aldosterone synthesis is mediated by type A natriuretic peptide receptors endowed with intrinsic guanylyl cyclase activity (29, 30), and cGMP was initially thought to be the intracellular messenger mediating all effects of ANP (29, 31). However, a number of studies reported that the inhibition of aldosterone synthesis by ANP could not be mimicked with membrane-permeant, nonhydrolysable analogs of cGMP, such as 8-bromo-cGMP or dibutyryl-cGMP (4, 32, 33). The intracellular sites of the antisteroidogenic action of ANP remain, therefore, to be elucidated.

In theory, ANP could impede the generation of the cytosolic Ca2+ ([Ca2+]c) signal and the resulting rise in mitochondrial free Ca2+ concentration ([Ca2+]m) that are observed upon challenge with Ang II and K+ (34). Alternatively, ANP could affect cholesterol supply to the mitochondria or intramitochondrial cholesterol transfer or interfere with either the expression or the action of the StAR protein (35, 36).

In the present study, we have examined whether ANP interferes with any of the above processes, using the Ca2+-clamp technique to mimic Ang II or K+ activation. We report that ANP inhibition is exerted on intramitochondrial cholesterol transfer and results from an inhibition of the Ca2+-induced synthesis of StAR protein and therefore of StAR protein accumulation within mitochondria. We provide evidence that the increase in StAR protein synthesis elicited by Ca2+ is a consequence of an increase in the steady state level of StAR mRNA, suggesting that Ca2+ activates StAR protein gene transcription and that ANP modulates this activation.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
ANP Inhibits Ca2+-Induced Pregnenolone and Aldosterone Biosynthesis
In Ca2+-clamped bovine glomerulosa cells, pregnenolone production was stimulated in a concentration-dependent manner by [Ca2+]c as previously reported (37). This activation was inhibited in a concentration-dependent manner by both Thr-Ala-Pro-Arg-human (h)ANP(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28) (urodilatin, an analog of ANP originally isolated from human urine) and hANP(4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28) (Fig. 1Go). Similar results were obtained for aldosterone production (data not shown). Both peptides were equipotent in preventing pregnenolone production in cells submitted to an intracellular calcium clamp of 600 nM, with an IC50 of approximately 1 nM and a maximal inhibition of 90% (Fig. 1BGo). They were therefore used indifferently in subsequent experiments. Since the Ca2+ signal generated by the high-Ca2+ clamp is, by definition, constant, these results globally suggested to us that the target for the inhibitory mechanism of ANP resides downstream of the production of the calcium signal.



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Figure 1. Effect of ANP on Ca2+-Induced Pregnenolone Formation in Bovine Adrenal Glomerulosa Cells

A, Glomerulosa cells were stimulated with a cytosolic Ca2+-clamp for 2 h, in the absence or in the presence of 10 nM urodilatin, as described in Materials and Methods. Pregnenolone production is expressed as a percentage of the production determined in control cells incubated in nominally Ca2+-free medium ([Ca2+]c < 100 nM). **, Significantly different (P < 0.01, n = 6) from the respective control without urodilatin. B, Concentration dependence of the inhibitory effect of hANP(4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 ) ({blacksquare}) and urodilatin (•) on pregnenolone production in high-Ca2+ (600 nM) clamped cells. Percent inhibition was calculated by comparing pregnenolone production above zero-Ca2+ control in the presence of ANP peptides to that measured in the absence of peptide. Each point is the mean ± SEM of two to nine separate experiments. Pregnenolone production: Control, 0.102 ± 0.02 nmol/mg prot/2 h; high-Ca2+, 0.319 ± 0.135; control + hANP (10 nM), 0.092 ± 0.037; high-Ca2+ + hANP, 0.120 ± 0.054.

 
The membrane-permeant analogs of cGMP, 8-Br-cGMP and dibutyryl-cGMP (10 µM), failed to mimick the antisteroidogenic action of ANP, as shown in Fig. 2Go. Ten-fold higher concentrations (100 µM) of the analogs were equally uneffective (data not shown).



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Figure 2. Lack of Effect of cGMP Analogs on Ca2+-Induced Pregnenolone Production

Glomerulosa cells were stimulated with a cytosolic Ca2+-clamp for 2 h, in the absence or in the presence of 8-Br-cGMP ({blacktriangleup}) or dibutyryl-cGMP (•) (10 µM), as described in Materials and Methods. Pregnenolone production is expressed as a percentage of the production determined in control cells incubated in nominally Ca2+-free medium ([Ca2+]c < 100 nM). Each point is the mean value of triplicate samples from two separate experiments.

 
ANP Does Not Affect Ang II-Induced [Ca2+]c and [Ca2+]m Responses
The lack of an effect of ANP on Ca2+ responses was confirmed in bovine glomerulosa cells transfected with targeted aequorin. In cells that had been pretreated with 100 nM hANP for 3–30 min, the [Ca2+]c response to Ang II in glomerulosa cells transfected with nontargeted-aequorin was not affected by the presence of hANP, and the [Ca2+]c values established under Ca2+ clamp were not altered (not shown). Since the mitochondrion is a known target for the Ca2+ signal, we also examined [Ca2+]m changes. When adrenal glomerulosa cells were challenged with Ang II (10 nM), a biphasic [Ca2+]m response was observed, as previously reported (34). In the presence of hANP, this response was superimposable to that recorded in the absence of hANP (Fig. 3Go). When added after Ang II challenge, hANP did not alter the plateau [Ca2+]m phase. Clearly, therefore, the generation of the calcium signal was unaffected by hANP.



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Figure 3. Lack of Effect of ANP on the [Ca2+]m Response to Ang II in Bovine Glomerulosa Cells

Cells were transfected with targeted aequorin as described in Materials and Methods. Aequorin luminescence was recorded under stimulation with Ang II (10 nM) in control cells (dotted trace) or in cells that had been pretreated with hANP (100 nM) (solid trace) for 30 min. This trace is representative of four similar experiments.

 
ANP Prevents Ca2+-Induced Stimulation of Intramitochondrial Cholesterol Transfer
We have previously shown that the stimulation of ionomycin-treated bovine adrenal glomerulosa cells with Ca2+ markedly decreased cholesterol content in outer mitochondrial membranes (OM) and concomitantly increased cholesterol content in contact sites (CS) and inner membranes (IM), reflecting a stimulation of intramitochondrial cholesterol transfer (35, 36). Since ANP almost entirely prevented Ca2+-supported pregnenolone formation, the first enzymatic step after cholesterol supply to P450scc, we therefore examined the effect of ANP on Ca2+-induced intramitochondrial cholesterol distribution. As we have previously shown, stimulation of glomerulosa cells with Ca2+ led to a significant increase of cholesterol content in CS and IM to 139.7 ± 10.7% and 131.5 ± 7% of the respective controls (P < 0.05, n = 3) and to a concomitant significant decrease of cholesterol content in OM, to 70.4% ± 2.1% of controls (P < 0.001, n = 3) (Fig. 4Go). hANP (10 nM) completely prevented this Ca2+-induced transfer of cholesterol to CS and IM (92.4 ± 4% and 103.1 ± 16.1% of the respective controls, n = 3). Moreover, no significant change was observed in cholesterol content in OM when glomerulosa cells were simultaneously treated with Ca2+ and hANP (88.2 ± 6.5% of controls, n = 3). hANP alone had no effect on cholesterol content in OM, CS, and IM (105.6 ± 2.9%, 95.5 ± 9.8%, and 104.2 ± 1.4% of the respective controls, n = 3) (Fig. 4Go).



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Figure 4. Effect of ANP on Ca2+-Induced Intramitochondrial Cholesterol Transfer in Bovine Glomerulosa Cells

Cells were stimulated with a cytosolic low- (<100 nM, control cells) or high- (600 nM) Ca2+ clamp for 2 h, in the absence or in the presence of 10 nM hANP(4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 ) as described in Materials and Methods. After submitochondrial fractionation, the cholesterol content of OM, CS, and IM in Ca2+ clamped cells was determined and expressed as a percentage of that measured in the respective submitochondrial fractions of control low-Ca2+ clamped cells (mean ± SEM, n = 3). In a typical experiment, mass unit values for cholesterol in OM, CS, and IM were, respectively, 10.9, 3.4, and 1.6 µg/mg protein for controls. (* and ***, significantly different from the respective control with P < 0.05 and P < 0.001, respectively).

 
ANP Prevents Ca2+-Induced Accumulation of StAR Protein within Mitochondria
We showed recently that Ca2+-activated intramitochondrial cholesterol transfer is accompanied in bovine glomerulosa cells by an increase in StAR protein within mitochondria, a finding consistent with a role for StAR protein in cholesterol transport (36). To determine whether ANP affects the StAR protein accumulation induced by Ca2+, we performed immunoblot analysis on mitochondrial proteins from glomerulosa cells that had been calcium-clamped in the presence or in the absence of hANP. As shown in Fig. 5Go, while Ca2+ induced the expected marked increase of StAR protein content in mitochondria (to 218.2 ± 44.4% of controls, n = 3, P < 0.01), hANP (10 nM) completely prevented this Ca2+-induced increase in StAR (94.5 ± 12.8% of controls). hANP had no significant effect in itself.



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Figure 5. Effect of ANP on the Ca2+-Induced Increase in StAR Protein Content in Mitochondria of Bovine Glomerulosa Cells

A, Mitochondria were isolated from low- or high-Ca2+ clamped cells incubated in the presence or in the absence of hANP (10 nM). Shown is a Western blot from one representative experiment. For each sample, 10 µg of mitochondrial proteins were analyzed by SDS-PAGE and immunoblotting as described in Materials and Methods. B, The immunospecific bands for StAR protein were quantitated by densitometry in three independent experiments. Control, low-Ca2+-clamp; Ca2+, high-Ca2+ clamp (600 nM); hANP, low-Ca2+ clamp in the presence of hANP (10 nM); Ca2+ + hANP, high-Ca2+ clamp in the presence of hANP. Integrated optical density (IOD) values are expressed as a percentage of that measured in mitochondria from control cells. **, Significantly different from control with P < 0.01, ++, significantly different from Ca2+ stimulation with P < 0.01.

 
ANP Prevents Ca2+-Induced Stimulation of StAR Protein Synthesis
The Ca2+-induced accumulation of StAR protein content in mitochondria could result from increased StAR protein synthesis. This hypothesis was tested in glomerulosa cells radiolabeled with [35S]methionine/cysteine during Ca2+ stimulation in the presence or in the absence of hANP. StAR protein was then immunoprecipitated from total cellular extracts as described in Materials and Methods. Upon SDS-PAGE analysis and autoradiography of StAR immunoprecipitates, we observed that, in high Ca2+-clamped cells, the labeling of StAR protein was significantly stimulated (167.5 ± 17.3% of controls, n = 3, P < 0.01) (Fig. 6Go), reflecting increased StAR protein synthesis. The addition of hANP (10 nM) simultaneously with Ca2+ completely prevented this increase in StAR protein labeling.



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Figure 6. Effect of ANP on the Ca2+-Induced Increase in StAR Protein Radiolabeling

A, Immunoprecipitation of StAR protein from [35S]methionine/cysteine-labeled bovine glomerulosa cells treated as described in the legend of Fig. 5Go. StAR protein was immunoprecipitated from equivalent amounts of radioactivity for each treatment as described in Materials and Methods, and the antibody-StAR protein complexes were analyzed by SDS-PAGE and autoradiography. B, Immunoprecipitated labeled StAR protein was quantitated by densitometry in three independent experiments. Integrated optical density (IOD) values are expressed as a percentage of that measured in control cells. **, Significantly different from control with P < 0.01; ++, significantly different from Ca2+ stimulation with P < 0.01.

 
ANP Inhibits Ca2+-Induced Increase in StAR mRNA Levels
The above results prompted us to examine whether ANP could exert its antisteroidogenic effect by acting directly on StAR mRNA production and/or stability. Upon Northern blot analysis of StAR mRNA, we observed consistently two, sometimes three, transcripts that hybridized with StAR cDNA. Two major bands migrated at approximately 2.6 and 1.6 kb (Fig. 7AGo), and a third one migrated at 0.9 kb. These transcripts showed coordinate induction. We therefore quantified only the most abundant 2.6-kb transcript. The quantification of StAR 2.6 kb mRNA from four independent experiments indicated that Ca2+ provoked a significant accumulation of this transcript (189 ± 14.5% of controls, P < 0.001) and that hANP (10 nM) prevented the Ca2+-induced increase in StAR mRNA (116 ± 13% of controls) (Fig. 7BGo).



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Figure 7. Effect of ANP on the Ca2+-Induced Increase in Steady State StAR mRNA Levels in Bovine Glomerulosa Cells

A, Northern blot analysis of StAR mRNA in control (C) and high-Ca2+ clamped cells, in the absence or in the presence of 10 nM hANP (lanes 1–3). In lanes 4–6, the same experiment was performed in the presence of actinomycin D (5 µg/ml). Total RNA was isolated as described in Materials and Methods and 15 µg of each sample were analyzed by agarose gel electrophoresis. StAR specific transcripts were detected by hybridization with a mouse full-length StAR cDNA. The blot was then stripped and rehybridized with a cDNA probe specific of the mouse GAPDH to normalize for equivalent loading of RNA. B, Quantification of StAR mRNA levels in four independent experiments (panel A, lanes 1–3). The major 2.6-kb transcript was quantitated and normalized to GAPDH mRNA levels. The corrected values for the IOD are expressed as a percentage of the IOD of StAR mRNA levels in control cells. ***, Significantly different from controls with P < 0.001; ++, significantly different from Ca2+ stimulation with P < 0.01 (n = 3). C, Quantification of StAR mRNA in control and high Ca2+-clamped cells incubated in the presence of actinomycin D, with or without hANP (10 nM) (panel A, lanes 4–6) (n = 3).

 
The Ca2+-induced accumulation of StAR mRNA may result from changes in transcription rate and/or in mRNA turnover. To determine whether Ca2+ or hANP affected StAR mRNA stability, glomerulosa cells were submitted to a high-calcium clamp (600–700 nM) for 2 h. The incubation buffer was then removed and replaced with fresh buffer containing ionomycin and actinomycin D. Incubation was then continued for 6 h in the presence or in the absence of Ca2+ and/or hANP (10 nM). After a 6 h-incubation period in Krebs buffer, StAR mRNA levels decayed to 69.6 ± 6.6% (n = 3) of the zero time value, whereas in the presence of Ca2+, StAR mRNA decayed to 52 ± 15% of the zero time value (n = 3). These differences were not statistically significant. The addition of hANP did not elicit any significant effect on StAR mRNA stability, as measured 6 h after the addition of actinomycin D (63.6 ± 4.7% of zero time value).

These results suggested that the effect of Ca2+ on the steady state levels of StAR mRNA must reflect transcriptional activation. To assess this possibility, glomerulosa cells were treated simultaneously with Ca2+ and actinomycin D (5 µg/ml), in the absence or in the presence of hANP (10 nM). Densitometric analysis of Northern blots from three independent experiments clearly indicated that actinomycin D completely prevented the Ca2+-induced StAR mRNA accumulation (108 ± 5% of controls) (Fig. 7CGo). In separate experiments, we observed that actinomycin D inhibited Ca2+-induced pregnenolone production by 69.8 ± 11.8% (n = 4). Since ANP had no effect on StAR mRNA stability, it is likely that ANP exerts its inhibitory effect on Ca2+-induced StAR mRNA accumulation by preventing StAR gene transcription.

Ca2+ Stimulates ANP-Sensitive Transcription of the StAR Gene
The above hypothesis was confirmed by the results of nuclear run-on experiments performed in bovine glomerulosa cells. As shown in Fig. 8Go, after 2 h of a high-Ca2+ clamp, transcriptional activity of the StAR gene was 171 ± 19% (P < 0.01, n = 3) of that measured in control (low-Ca2+ clamp) cells. Once again, hANP (10 nM) prevented this Ca2+-induced activation of StAR gene transcription. Transcription of the ß-actin gene was not significantly affected by Ca2+ or hANP, and no signal was detected with the empty vector (Fig. 8AGo).



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Figure 8. Nuclear Run-on Transcriptional Analysis of the StAR Gene

A, Bovine glomerulosa cells were submitted to a low- (control) or a high-Ca2+ (600 nM) clamp in the presence or in the absence of hANP (10 nM), as described in Materials and Methods. The transcription of the StAR gene was assessed in isolated nuclei incubated with [32P]UTP to generate radiolabeled RNA transcripts. RNA was hybridized to membranes on which StAR cDNA cloned in a Bluescript plasmid, empty Bluescript plasmid (pBSK, negative control), and mouse ß-actin cDNA cloned in Bluescript (invariant internal control) had been immobilized. B, Quantification of nuclear run-on analysis from three independent experiments. Results are expressed as a percentage of controls.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
ANP exerts one of its major physiological antihypertensive effects by preventing the stimulation of aldosterone secretion elicited in adrenal glomerulosa cells by agonists such as Ang II and K+ (4, 26, 38). Although ANP is known to stimulate cGMP formation in glomerulosa cells (29, 31), the intracellular mechanism of action of ANP as well as its molecular targets in adrenocortical cells are still poorly defined, due in part to the failure of membrane-permeant, nonhydrolysable analogs of cGMP to mimic the biological effect of ANP (4, 32, 33). This lack of effect of cGMP analogs was also observed in this work.

The present study was therefore undertaken in an attempt to identify intracellular targets for the inhibitory action of ANP on aldosterone biosynthesis. In a previous work on the mechanisms of activation of aldosterone biosynthesis by Ang II and Ca2+ in bovine adrenal glomerulosa cells, we have shown that physiological rises in [Ca2+]c acutely stimulate the rate-limiting step in this process, namely cholesterol translocation from the mitochondrial outer membrane to intermembrane contact sites and inner membrane (35). Concomitantly, Ca2+ induced a specific increase in StAR protein in the inner mitochondrial membrane, confirming the well documented role of StAR in intramitochondrial cholesterol redistribution (36). In addition, both Ca2+-stimulated intramitochondrial cholesterol transfer and StAR accumulation in the inner membrane were cycloheximide-sensitive, indicating the requirement for ongoing protein synthesis in the acute activation of aldosterone production. Using the ionomycin-mediated calcium clamp technique as a tool to mimic agonist-induced physiological rises of [Ca2+]c (37), we have investigated whether ANP affects any of these Ca2+-sensitive events.

Two major conclusions can be drawn from the results of the present work: 1) Ca2+ regulates StAR gene expression at the transcriptional level and 2) ANP inhibits aldosterone production by preventing Ca2+-induced StAR mRNA accumulation.

While the role of cAMP in mediating the activation of StAR gene transcription via steroidogenic factor 1 (SF-1), an orphan nuclear receptor, has been extensively investigated (39, 40, 41, 42, 43), the potential participation of Ca2+ in regulating StAR gene expression has received little attention to date. We first examined the effect of Ca2+ stimulation on StAR mRNA levels. In agreement with previous data on bovine StAR gene expression (43), Northern blot analysis of StAR mRNA showed that two major StAR mRNA transcripts of 2.6 and 1.6 kb were present in bovine adrenal cells. Importantly, Ca2+ induced a marked and significant increase in StAR mRNA levels within 2 h of exposure of glomerulosa cells to a high Ca2+-clamp. This observation raised the question of whether Ca2+ could increase StAR mRNA half-life. Our results show that StAR mRNA decayed to the same extent in low- and high-Ca2+ clamped cells for up to 6 h after addition of actinomycin D, thus excluding a stabilizing effect as the mechanism leading to Ca2+-induced acute accumulation of StAR mRNA.

Rather, the clear-cut inhibitory effect of actinomycin D on the Ca2+-induced increase in StAR mRNA steady state levels suggested a direct relationship between StAR gene transcription and Ca2+-stimulated pregnenolone production. However, data from the literature on the requirement of transcription for the acute steroidogenic response are conflicting. Indeed, actinomycin D exerted little or no effect on ACTH-induced RNA synthesis and corticosteroid production in rat adrenal quarters or dispersed cells (44, 45), while it blocked to a variable extent RNA synthesis and steroid production in bovine adrenal slices (44), in a mouse testicular interstitial cell line (46), in rat testicular interstitial cells in primary culture, and in Leydig cells (47, 48). More recently, Clark et al. (49) demonstrated that actinomycin D potently inhibits StAR mRNA accumulation and steroid production in MA-10 mouse Leydig tumor cells. Our results with bovine glomerulosa cells are consistent with the studies on testicular cells, MA-10 cells, and, more importantly, bovine adrenal slices and strongly speak in favor of a transcriptional activation exerted by Ca2+.

Increased [Ca2+]c up-regulates eukaryotic gene and, in particular, immediate-early response gene transcription in a variety of cell types (50). In rat aortic smooth muscle cells (51), as well as in bovine adrenal glomerulosa (52) and fasciculata (53) cells, Ang II was found to induce the expression of the early response genes c-jun, c-fos and/or Jun B. Whether these protooncogenes could be involved in the control of StAR gene transcription remains to be determined. However, at the present time, no activator protein-1 (AP-1)-responsive elements have been found in the 1.3 kb of DNA upstream of the transcription start site (39, 41). Interestingly, Sugawara et al. (39) reported the presence within the StAR promoter of two Sp1-responsive elements. This transcription factor has recently been shown to mediate Ca2+-regulated gene expression in various cell types (50, 54). Alternatively, StAR gene transcription could be promoted by a Ca2+-dependent relief of a blockade to elongation in intragenic sites, as has been proposed for c-fos transcription (50). Whatever the mechanism, our results clearly demonstrate a dual site of action for the calcium messenger in the acute activation of mineralocorticoid biosynthesis: in addition to its previously described intramitochondrial effects (35, 36, 37, 55), Ca2+ exerts clear-cut rapid genomic actions that result in de novo synthesis of StAR protein.

The second important finding of this work relates to the antisteroidogenic action of ANP. While ANP did not affect the [Ca2+]c and [Ca2+]m changes elicited by either Ang II or a Ca2+ clamp, it markedly reduced the Ca2+-supported increase in pregnenolone formation, indicating that the inhibition occurred downstream of the generation of the Ca2+ signal. By contrast, ANP did not significantly affect pregnenolone synthesis in control nonstimulated cells. These results are consistent with previous studies suggesting that ANP may interfere with the early steps of steroidogenesis (22, 23, 24, 56). Elliott and Goodfriend (23) reported that the inhibitory effect of ANP on Ang II-elicited aldosterone synthesis was less effective at high extracellular Ca2+ concentrations, suggesting that ANP may impair a process that is stimulated by Ca2+. Our data demonstrate for the first time that ANP decreases the availability of endogenous cholesterol to the P450scc enzyme, by preventing Ca2+-stimulated intramitochondrial cholesterol transfer. Earlier studies on the hormonal regulation of cholesterol mobilization in steroidogenic cells had demonstrated that ongoing synthesis of StAR protein is required to facilitate cholesterol translocation to the inner mitochondrial membrane. Our results show that ANP completely abolished both the accumulation of mature StAR protein within mitochondria and the increase in [35S]methionine labeling of StAR protein in high Ca2+-clamped glomerulosa cells. This finding indicates that ANP inhibits the Ca2+-induced de novo synthesis of StAR protein and is consistent with previous data reported by Elliott et al. (57). Using two-dimensional gel electrophoresis, these authors have shown that Ang II induces in bovine adrenal glomerulosa cells the rapid appearance of a family of 28- to 30-kDa proteins, subsequently assumed to be StAR proteins (58), and that cotreatment with ANP and Ang II markedly reduced the amount of these proteins in mitochondria.

Interestingly, ANP abolished the Ca2+-elicited increase in StAR mRNA levels. We therefore examined whether this inhibitory effect of ANP was due to blockade of StAR gene transcription and/or to a decrease in StAR mRNA stability. RNA decay experiments performed in the presence of actinomycin D showed that ANP did not alter StAR mRNA stability. Hence, it was likely that ANP counteracted the positive effect of Ca2+ on StAR transcription. Further evidence for the transcriptional control of the StAR gene by Ca2+ and ANP was obtained using nuclear run-on assays. To our knowledge, this is the first report relating increases in intracellular Ca2+ to transcriptional induction of StAR gene as well as an inhibitory effect of ANP on this process. The process by which ANP exerts its negative control through non-cGMP-mediated mechanisms remains to be elucidated. Interestingly, DAX-1, a member of the nuclear-receptor superfamily of transcription factors, has been recently shown to repress StAR gene expression and steroidogenesis (59), and one could speculate that ANP might induce DAX-1 expression in adrenal glomerulosa cells.

In summary, our results directly show that Ca2+ ion is a potent physiological regulator of StAR gene expression in bovine adrenal glomerulosa cells and that ANP impedes this transcriptional activation, thus exerting its physiological inhibitory action on aldosterone biosynthesis.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Chemicals
Ionomycin was purchased from Calbiochem (Lucerne, Switzerland). Human ANP [hANP(4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28)] and urodilatin [Thr-Ala-Pro-Arg-hANP(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28)] were obtained from Bachem (Bubendorf, Switzerland). [3H]pregnenolone was purchased from Amersham (Zurich, Switzerland), [{alpha}-32P]dCTP (3000 Ci/mmol) and [{alpha}-32P]UTP (800 Ci/mmol) from Hartmann Analytic (Braunschweig, Germany). [35S]methionine/cysteine labeling mix was purchased from ICN Biomedicals GmbH (Eschwege, Germany). Antipregnenolone antiserum was obtained from Biogenesis Ltd (Poole, UK). Win 19758 was purchased from Farillon (London, UK). All other chemicals used were purchased from Sigma (St. Louis, MO) or from Fluka (Buchs, Switzerland).

Bovine Adrenal Zona Glomerulosa Cell Culture and Treatments
Bovine adrenal glands were obtained from a local slaughterhouse. Zona glomerulosa cells were prepared by enzymatic dispersion with dispase and purified on Percoll density gradients (60). Primary cultures of purified glomerulosa cells were established as described in detail elsewhere (60) and kept in serum-free medium for 1 day before experiments, which were performed on the third day of culture.

Cells cultured in 10 cm plastic Petri dishes (107 cells per dish) or in 24-well plates (500,000 cells per well) were washed twice with a modified Krebs-Ringer (NaCl, 136 mM; NaHCO3, 5 mM; KH2PO4, 1.2 mM; MgSO4, 1.2 mM; KCl, 1.8 mM; EGTA, 0.2 mM; D-glucose, 5.5 mM; HEPES, 20 mM; pH 7.4) and preincubated at 37 C for 30 min in the same buffer. Ca2+-clamping was performed at 37 C for 2 h, in the presence of 2 µM ionomycin, 1 mM total extracellular Ca2+, and 0.2 mM EGTA, to achieve a [Ca2+]c of 600–700 nM (high-Ca2+ clamp), as described elsewhere (37). Control cells were Ca2+-clamped in Krebs-Ringer buffer lacking Ca2+, in the presence of 0.2 mM EGTA (low-Ca2+ clamp, [Ca2+]c < 100 nM). Thr-Ala-Pro-Arg-hANP(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28), ANP(4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28), or membrane-permeant cGMP analogs were added to the incubation medium during stimulation with Ca2+ as indicated. In experiments designed to measure cholesterol content in submitochondrial membranes, aminogluthetimide (500 µM) was included in the incubation medium to inhibit cholesterol side-chain cleavage. At the end of the incubation period, the cells were scraped and sedimented at 200 x g for 15 min before subcellular fractionation or processing for total RNA isolation as described hereafter.

Steroid Measurement
Aldosterone content in incubation media was measured by direct RIA, using a commercially available kit (Diagnostic Systems Laboratories, Webster, TX). For the assessment of pregnenolone production, WIN 19758 (5 µM) was included in the incubation medium to prevent conversion of pregnenolone into progesterone. At the end of the incubation period, pregnenolone was determined directly in the medium by RIA. Steroid production was normalized and expressed per milligram cellular protein.

Measurement of [Ca2+]c and [Ca2+]m
[Ca2+]m was measured in bovine glomerulosa cells transfected with a plasmid coding for mitochondrial matrix-targeted aequorin as previously described in detail (34). [Ca2+]c was determined in cells transfected with the same plasmid lacking the targeting mitochondrial presequence cDNA. Cells were transfected by the phosphate calcium precipitation method using the CellPhect Transfection Kit (Pharmacia Biotech, Zürich, Switzerland), according to the specifications of the manufacturer.

Preparation of Submitochondrial Fractions and Cholesterol Measurements
Glomerulosa cells were homogenized with a Potter-Elvejhem homogenizer (1200 rpm, 35 strokes), in a 5 mM Tris-HCl buffer, pH 7.4, containing 275 mM sucrose. The homogenate was centrifuged at 200 x g for 15 min to remove large debris and nuclei. Further centrifugation of the supernatant at 10,000 x g for 10 min yielded the mitochondria. The mitochondrial pellet was washed twice at 8,000 x g with the same buffer. Osmotically shocked adrenocortical mitochondria were fractionated on a sucrose density gradient into OM, CS, and IM as previously described (35). Protein was quantified using the Bio-Rad protein microassay (Bio-Rad Laboratories, Richmond, CA) and BSA as a standard. The cholesterol content of submitochondrial fractions (15–20 µg protein for OM, 30–40 µg protein for CS, and 80–100 µg for IM) was determined by a coupled cholesterol oxidase-peroxidase assay as reported elsewhere (35).

SDS-PAGE
SDS-PAGE was performed according to Laemmli (61). Mitochondrial proteins (10 µg/lane) were solubilized in sample buffer (60 mM Tris-HCl, pH 6.8, 2% SDS, 5% ß-mercaptoethanol, 10% glycerol, 0.01% bromophenol blue) and loaded onto a 12% SDS-PAGE minigel (Mini Protean II System, Bio-Rad). Electrophoresis was performed at 150 V for 1 h.

Blotting Procedure and Immunodetection
SDS-PAGE-resolved proteins were electrophoretically transferred onto a nitrocellulose membrane (Schleicher & Schuell, Feldbach, Switzerland) according to Towbin et al. (62). After transfer, the membrane was incubated in a blocking buffer (PBS buffer containing 0.4% Tween 20 and 5% nonfat dry milk) for 1 h at room temperature, and then incubated with an antiserum generated by CovalAb (Lyon, France) against a peptide fragment (amino acids 88–98) of the 30- kDa StAR for 1 h in PBS/Tween 20 buffer (36). The membrane was thoroughly washed with the same buffer (3 x 10 min), and then incubated for 1 h with horseradish peroxidase-labeled goat antirabbit IgG (CovalAb). The nitrocellulose sheet was then washed four times for 15 min, and the antigen-antibody complex was revealed by enhanced chemiluminescence, using the Western blotting detection kit and Hyper-ECL film from Amersham.

Radiolabeling of Glomerulosa Cells and Immunoprecipitation of StAR
For radiolabeling of cellular proteins, 3 x 106 glomerulosa cells, plated in 6-cm petri dishes, were calcium-clamped for 2 h at 37 C, in the presence or in the absence of ANP. The incubation medium (3 ml) contained [35S]methionine-cysteine (300 µCi). At the end of the labeling period, cells were washed three times with ice-cold PBS and lysed in RIPA buffer (10 mM sodium phosphate, pH 7.4, containing 150 mM NaCl, 1% Triton, 1% sodium deoxycholate, 1 mM phenylmethylsulfonylfluoride, 1 µg/ml aprotinin, and 1 µg/ml leupeptin). The lysate was cleared by centrifugation for 5 min at 12,000 x g at 4 C. The StAR antiserum was added at a 1:100 dilution to aliquots of the supernatant (200 µl, ~100 µg protein), which were then gently rocked for 2 h at 4 C, before being incubated for 30 min with Protein-A Sepharose beads (Pharmacia Biotech AG, Dubendorf, Switzerland). Immunoprecipitates were pelleted, washed four times with RIPA buffer and analyzed by SDS-PAGE and autoradiography.

RNA Isolation and Northern Blot Analysis
Glomerulosa cell total RNA was extracted using the RNAgents kit (Promega, Zurich, Switzerland) according to the instructions of the manufacturer. This system consistently yields 50–80 µg total RNA/107 cells. For Northern blot analysis, 15–20 µg RNA were size-fractionated on a 1% formaldehyde agarose gel, vacuum-transferred onto Nytran membranes (Schleicher & Schuell) and fixed by UV cross-linking. The integrity of the 18 S and 28 S RNA was checked by ethidium bromide staining of the gel. Hybridization was performed using the previously cloned 1.5-kb mouse StAR cDNA (16). The cDNA was labeled with [{alpha}32P]dCTP using the Rediprime random primer labeling kit from Amersham. Northern blots were prehybridized in Rapid Hybridization Buffer (Amersham) at 65 C for 30 min. The {alpha}32P-labeled probe (specific activity: 2 x 106 cpm/ng DNA) was then added and the incubation was continued for 2 h at 65 C. Blots were washed for 5 min and 15 min successively at room temperature in 2 x saline sodium citrate (SSC), 0.1% SDS, and then for 15 min in 1 x SSC, 0.1% SDS. The final wash was performed at 65 C for 15 min in 1 x SSC, 0.1% SDS. RNA-cDNA hybrids were visualized on Hyperfilms (Amersham) after a 12- to 24-h exposure period. Blots were stripped and reprobed with mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA (Ambion, Lugano, Switzerland) to assess RNA loading.

Nuclear Run-on Assays
Nuclear run-on was performed according to described protocols (63). Bovine glomerulosa cells (5 x 107 cells per treatment) were subjected to a low- or high-calcium clamp in the presence or in the absence of hANP for 2 h. Transcription reactions were then carried out on isolated nuclei using 150 µCi 32P-labeled UTP and 1 mM ATP, CTP, and GTP (Pharmacia Biotech AG, Dübendorf, Switzerland) in reaction buffer (5 mM Tris-HCl, pH 8, 2.5 mM MgCl2, 150 mM KCl) at 30 C for 30 min. Nuclear RNA was isolated and hybridized for 36 h at 65 C to Hybond-N+-charged nylon membranes (Amersham) on which 5 µg of a Bluescript plasmid containing a 1.5-kb StAR cDNA insert, 5 µg of a Bluescript plasmid containing a 250-bp mouse ß-actin insert (Ambion), or 5 µg of empty Bluescript plasmid had been immobilized. Membranes were washed and exposed to hyperfilms for visualization.

Analysis of Data
Results are expressed as means ± SEM. The mean values were compared by ANOVA using Fisher’s test. A value of P < 0.05 was considered as statistically significant. Quantification of immunoblots and autoradiograms was performed using a Molecular Dynamics (Sunnyvale, CA) Computing Densitometer.


    ACKNOWLEDGMENTS
 
The authors are grateful to Liliane Bockhorn, Walda Dimeck, and Gisèle Dorenter for their excellent technical assistance.


    FOOTNOTES
 
Address requests for reprints to: Dr. Nadia Cherradi, Division of Endocrinology and Diabetology, University Hospital, 24 rue Micheli-du-Crest, CH-1211 Geneva 14, Switzerland. E-mail: nadia.cherradi{at}diogenes.hcuge.ch

This work was supported in part by Swiss National Science Foundation Grants 31.42178–94 (to A.M.C.) and 32.49297.96 (to M.F.R.), by NIH Grant HD-17841 (to D.M.S.) and by grants from the Ciba-Geigy Jubiläumsstiftung and the Sandoz Foundation. M.F.R. is a recipient of a grant from the Professor Max Cloëtta Foundation.

Received for publication January 16, 1998. Revision received March 11, 1998. Accepted for publication March 17, 1998.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Capponi AM, Lew PD, Jornot L, Vallotton MB 1984 Correlation between cytosolic free Ca2+ and aldosterone production in bovine adrenal glomerulosa cells. Evidence for a difference in the mode of action of angiotensin II and potassium. J Biol Chem 259:8863–8869[Abstract/Free Full Text]
  2. Müller J Regulation of aldosterone biosynthesis. 1988 Monographs in Endocrinology. Springer-Verlag, Berlin
  3. Quinn SJ, Williams GH, Tillotson DL 1988 Calcium response of single adrenal glomerulosa cells to external potassium. Am J Physiol 255:E488–E495
  4. Barrett PQ, Bollag WB, Isales CM, McCarthy RT, Rasmussen H 1989 Role of calcium in angiotensin II-mediated aldosterone secretion. Endocr Rev 10:496–518[Medline]
  5. Spät A, Enyedi P, Hajnoczky G, Hunyady L 1991 Generation and role of calcium signal in adrenal glomerulosa cells. Exp Physiol 76:859–885[Medline]
  6. Crivello JF, Jefcoate CR 1980 Intracellular movement of cholesterol in rat adrenal cells. Kinetics and effects of inhibitors. J Biol Chem 255:8144–8155[Free Full Text]
  7. Jefcoate CR, McNamara BC, Artemenko I, Yamazaki T 1992 Regulation of cholesterol movement to mitochondrial cytochrome P450scc in steroid hormone synthesis. J Steroid Biochem Mol Biol 43:751–767[CrossRef]
  8. Privalle CT, Crivello JF, Jefcoate CR 1983 Regulation of intramitochondrial cholesterol transfer to side-chain cleavage cytochrome P-450 in rat adrenal gland. Proc Natl Acad Sci USA 80:702–706[Abstract]
  9. Privalle CT, McNamara BC, Dhariwal MS, Jefcoate CR 1987 ACTH control of cholesterol side-chain cleavage at adrenal mitochondrial cytochrome P-450scc. Regulation of intramitochondrial cholesterol transfer. Mol Cell Endocrinol 53:87–101[CrossRef][Medline]
  10. Orme-Johnson NR 1990 Distinctive properties of adrenal cortex mitochondria. Biochim Biophys Acta 1020:213–231[Medline]
  11. Elliott ME, Goodfriend TL 1984 Identification of the cycloheximide-sensitive site in angiotensin-stimulated aldosterone synthesis. Biochem Pharmacol 33:1519–1524[CrossRef][Medline]
  12. Alberta JA, Epstein LF, Pon LA, Orme-Johnson NR 1989 Mitochondrial localization of a phosphoprotein that rapidly accumulates in adrenal cortex cells exposed to adrenocorticotropic hormone or to cAMP. J Biol Chem 264:2368–2372[Abstract/Free Full Text]
  13. Epstein LF, Orme-Johnson NR 1991 Regulation of steroid hormone biosynthesis. Identification of precursors of a phosphoprotein targeted to the mitochondrion in stimulated rat adrenal cortex cells. J Biol Chem 266:19739–19745[Abstract/Free Full Text]
  14. Stocco DM, Sodeman TC 1991 The 30-kDa mitochondrial proteins induced by hormone stimulation in MA-10 mouse Leydig tumor cells are processed from larger precursors. J Biol Chem 266:19731–19739[Abstract/Free Full Text]
  15. Stocco DM 1992 Further evidence that the mitochondrial proteins induced by hormone stimulation in MA-10 mouse Leydig tumor cells are involved in the acute regulation of steroidogenesis. J Steroid Biochem Mol Biol 43:319–333[CrossRef][Medline]
  16. Clark BJ, Wells J, King SR, Stocco DM 1994 The purification, cloning and expression of a novel luteinizing hormone-induced mitochondrial protein in MA-10 mouse Leydig tumor cells. Characterization of the steroidogenic acute regulatory protein (StAR). J Biol Chem 269:28314–28322[Abstract/Free Full Text]
  17. King SR, Ronen-Fuhrmann T, Timberg R, Clark BJ, Orly J, Stocco DM 1995 Steroid production after in vitro transcription, translation, and mitochondrial processing of protein products of complementary deoxyribonucleic acid for steroidogenic acute regulatory Protein. Endocrinology 136:5165–5176[Abstract]
  18. Stocco DM, Clark BJ 1996 Role of the steroidogenic acute regulatory protein (StAR) in steroidogenesis. Biochem Pharmacol 51:197–205[CrossRef][Medline]
  19. Stocco DM, Clark BJ 1996 Regulation of the acute production of steroids in steroidogenic cells. Endocr Rev 17:221–244[Medline]
  20. Lin D, Sugawara T, Strauss JF, Clark BJ, Stocco DM, Saenger P, Rogol A, Miller WL 1995 Role of steroidogenic acute regulatory protein in adrenal and gonadal steroidogenesis. Science 267:1828–1831[Medline]
  21. Bose HS, Sugawara T, Strauss JF, Miller WL 1996 The pathophysiology and genetics of congenital lipoid adrenal hyperplasia. N Engl J Med 335:1870–1878[Abstract/Free Full Text]
  22. Racz K, Kuchel O, Cantin M, De Léan A 1985 Atrial natriuretic factor inhibits the early pathway of steroid biosynthesis in bovine adrenal cortex. FEBS Lett 192:19–22[CrossRef][Medline]
  23. Elliott ME, Goodfriend TL 1986 Inhibition of aldosterone synthesis by atrial natriuretic factor. Fed Proc 45:2376–2381[Medline]
  24. Isales CM, Bollag WB, Kiernan LC, Barrett PQ 1989 Effect of ANP on sustained aldosterone secretion stimulated by angiotensin II. Am J Physiol 256:C89–C95
  25. Nawata H, Ohashi M, Haji M, Takayanagi R, Higuchi K, Fujio N, Hashiguchi T, Ogo A, Nakao R, Ohnaka K, Nishi Y 1991 Atrial and brain natriuretic peptide in adrenal steroidogenesis. J Steroid Biochem Mol Biol 40:367–379[CrossRef][Medline]
  26. Goodfriend TL, Elliott ME, Atlas SA 1984 Actions of synthetic atrial natriuretic factor on bovine adrenal glomerulosa. Life Sci 35:1675–1682[CrossRef][Medline]
  27. Chartier L, Schiffrin EL, Thibault G, Garcia R 1984 Atrial natriuretic factor inhibits the stimulation of aldosterone secretion by angiotensin II, ACTH and potassium in vitro and angiotensin II-induced steroidogenesis in vivo. Endocrinology 115:2026–2028[Abstract]
  28. Kudo T, Baird A 1984 Inhibition of aldosterone production in the adrenal glomerulosa by atrial natriuretic factors. Nature 312:756–757[Medline]
  29. Hamet P, Tremblay J, Pang SC, Garcia R, Thibault G, Gutkowska J, Cantin M, Genest J 1984 Effect of native and synthetic atrial natriuretic factor on cyclic GMP. Biochem Biophys Res Commun 123:515–527[Medline]
  30. Drewett JG, Garbers DL 1994 The family of guanylyl cyclase receptors and their ligands. Endocr Rev 15:135[Medline]
  31. Waldman SA, Rapoport RM, Murad F 1984 Atrial natriuretic factor selectively activates particulate guanylate cyclase and elevates cyclic GMP in rat tissues. J Biol Chem 259:14332–14334[Abstract/Free Full Text]
  32. Barrett PQ, Isales CM 1988 The role of cyclic nucleotides in atrial natriuretic peptide-mediated inhibition of aldosterone secretion. Endocrinology 122:799–808[Abstract]
  33. Isales CM, Lewicki JA, Nee JJ, Barrett PQ 1992 ANP-(7–23) stimulates a DHP-sensitive Ca-2+ conductance and reduces cellular cAMP via a cGMP-independent mechanism. Am J Physiol 263:C334–C342
  34. Brandenburger Y, Kennedy ED, Python CP, Rossier MF, Vallotton MB, Wollheim CB, Capponi AM 1996 Possible role for mitochondrial calcium in angiotensin II- and potassium-stimulated steroidogenesis in bovine adrenal glomerulosa cells. Endocrinology 137:5544–5551[Abstract]
  35. Cherradi N, Rossier MF, Vallotton MB, Capponi AM 1996 Calcium stimulates intramitochondrial cholesterol transfer in bovine adrenal glomerulosa cells. J Biol Chem 271:25971–25975[Abstract/Free Full Text]
  36. Cherradi N, Rossier MF, Vallotton MB, Timberg R, Friedberg I, Orly J, Wang XJ, Stocco DM, Capponi AM 1997 Submitochondrial distribution of three key steroidogenic proteins (steroidogenic acute regulatory protein, P450 side-chain cleavage and 3ß-hydroxysteroid dehydrogenase isomerase enzymes) upon stimulation by intracellular calcium in adrenal glomerulosa cells. J Biol Chem 272:7899–7907[Abstract/Free Full Text]
  37. Python CP, Laban OP, Rossier MF, Vallotton MB, Capponi AM 1995 The site of action of Ca2+ in the activation of steroidogenesis: studies in Ca2+-clamped bovine adrenal zona-glomerulosa cells. Biochem J 305:569–576[Medline]
  38. Itoh H, Nakai K, Katsuura G, Morii N, Sioko SS, Sakamoto M, Sugawara A, Yamada T, Saito Y, Matsushito A, Imura H 1986 Centrally infused atrial natriuretic peptide attenuates exaggerated salt appetite in spontaneously hypertensive rats. Circ Res 59:342–347[Abstract]
  39. Sugawara T, Holt JA, Kiriakidou M, Strauss JF 1996 Steroidogenic factor 1-dependent promoter activity of the human steroidogenic acute regulatory protein (StAR) gene. Biochemistry 35:9052–9059[CrossRef][Medline]
  40. Caron KM, Ikeda Y, Soo SC, Stocco DM, Parker KL, Clark BJ 1997 Characterization of the promoter region of the mouse gene encoding the steroidogenic acute regulatory protein. Mol Endocrinol 11:138–147[Abstract/Free Full Text]
  41. Sugawara T, Kiriakidou M, Mcallister JM, Holt JA, Arakane F, Strauss JF 1997 Regulation of expression of the steroidogenic acute regulatory protein (StAR) gene: A central role for steroidogenic factor 1. Steroids 62:5–9[CrossRef][Medline]
  42. Sugawara T, Kiriakidou M, Mcallister JM, Kallen CB, Strauss JF 1997 Multiple steroidogenic factor 1 binding elements in the human steroidogenic acute regulatory protein gene 5'-flanking region are required for maximal promoter activity and cyclic AMP responsiveness. Biochemistry 36:7249–7255[CrossRef][Medline]
  43. Pilon N, Daneau I, Brisson C, Ethier JF, Lussier JG, Silversides DW 1997 Porcine and bovine steroidogenic acute regulatory protein (StAR) gene expression during gestation. Endocrinology 138:1085–1091[Abstract/Free Full Text]
  44. Farese RV 1966 Effects of actinomycin D on ACTH-induced corticosteroidogenesis. Endocrinology 78:929–936[Medline]
  45. Schulster D 1974 Corticosteroid and ribonucleic acid synthesis in isolated adrenal cells: inhibition by actinomycin D. Mol Cell Endocrinol 1:55–64[CrossRef]
  46. Shin SI, Sato GH 1971 Inhibition by actinomycin D, cycloheximide and puromycin of steroid synthesis induced by cyclic AMP in interstitial cells. Biochem Biophys Res Commun 45:501–504[Medline]
  47. Mendelson C, Dufau M, Catt KJ 1975 Dependence of gonadotropin-induced steroidogenesis upon RNA and protein synthesis in the interstitial cells of the rat testis. Biochim Biophys Acta 411:222–230[Medline]
  48. Cooke BA, Janszen FHA, van Driel MJA, van der Molen HJ 1979 Evidence for the involvement of lutropin-independent RNA synthesis in Leydig cell steroidogenesis. Mol Cell Endocrinol 14:181–189[CrossRef][Medline]
  49. Clark BJ, Combs R, Hales KH, Hales DB, Stocco DM 1997 Inhibition of transcription affects synthesis of steroidogenic acute regulatory protein and steroidogenesis in MA-10 mouse Leydig tumor cells. Endocrinology 138:4893–4901[Abstract/Free Full Text]
  50. Roche E, Prentki M 1994 Calcium regulation of immediate-early response genes. Cell Calcium 16:331–338[Medline]
  51. Taubman MB, Berk BC, Izumo S, Tsuda T, Alexander RW, Nadal-Ginard B 1989 Angiotensin II induces c-fos mRNA in aortic smooth muscle. Role of Ca++ mobilization and protein kinase C activation. J Biol Chem 264:526–530[Abstract/Free Full Text]
  52. Clark AJL, Balla T, Jones MR, Catt KJ 1992 Stimulation of early gene expression by angiotensin II in bovine adrenal glomerulosa cells. Roles of calcium and protein kinase C. Mol Endocrinol 6:1889–1898[Abstract]
  53. Viard I, Penhoat A, Ouali R, Langlois D, Bégeot M, Saez JM 1994 Peptide hormone and growth factor regulation of nuclear proto-oncogenes and specific functions in adrenal cells. J Steroid Biochem Mol Biol 50:219–224[CrossRef][Medline]
  54. McDonough PM, Hanford DS, Sprenkle AA, Mellon NR, Glembotski CC 1997 Collaborative roles for c-Jun N-terminal kinase, c-Jun, Serum response factor, and Sp1 in calcium-regulated myocardial gene expression. J Biol Chem 272:24046–24053[Abstract/Free Full Text]
  55. Capponi AM, Rossier MF, Davies E, Vallotton MB 1988 Calcium stimulates steroidogenesis in permeabilized bovine adrenal cortical cells. J Biol Chem 263:16113–16117[Abstract/Free Full Text]
  56. Apfeldorf WJ, Isales CM, Barrett PQ 1988 Atrial natriuretic peptide inhibits the stimulation of aldosterone secretion but not the transient increase in intracellular free calcium concentration induced by angiotensin II addition. Endocrinology 122:1460–1465[Abstract]
  57. Elliott ME, Goodfriend TL, Jefcoate CR 1993 Bovine adrenal glomerulosa and fasciculata cells exhibit 28.5-kilodalton proteins sensitive to angiotensin, other agonists, and atrial natriuretic peptide. Endocrinology 133:1669–1677[Abstract]
  58. Elliott ME, Goodfriend TL, Ball DL, Jefcoate CR 1997 Angiotensin-responsive adrenal glomerulosa cell proteins: characterization by protease mapping, species comparison, and specific angiotensin receptor antagonists. Endocrinology 138:2530–2536[Abstract/Free Full Text]
  59. Zazopoulos E, Lalli E, Stocco D, Sassone-Corsi P 1997 DNA binding and transcriptional repression by DAX-1 blocks steroidogenesis. Nature 390:311–315[CrossRef][Medline]
  60. Python CP, Rossier MF, Vallotton MB, Capponi AM 1993 Peripheral-type benzodiazepines inhibit calcium channels and aldosterone production in adrenal glomerulosa cells. Endocrinology 132:1489–1496[Abstract]
  61. Laemmli UK 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 27:680–685
  62. Towbin H, Staehelin T, Gordon J 1979 Electrophoretic transfer of proteins from polyacrylamide gel to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76:4350–4354[Abstract]
  63. Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (eds) 1994 Current Protocols in Molecular Biology. John Wiley, New York