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Endocrinology Vol. 144, No. 8 3376-3381
Copyright © 2003 by The Endocrine Society

Corticosterone Enhances Adrenocorticotropin-Induced Calcium Signals in Bovine Adrenocortical Cells

Tomoko Chiyo, Takeshi Yamazaki, Kenji Aoshika, Shiro Kominami and Yoshihiro Ohta

Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Nakacho, Koganei, Tokyo, 184-8588, Japan; and Faculty of Integrated Arts and Sciences, Hiroshima University (T.Y., S.K.), Higashihiroshima 739-8521, Japan

Address all correspondence and requests for reprints to: Dr. Yoshihiro Ohta, Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Nakacho, Koganei, Tokyo 184-8588, Japan. E-mail: ohta{at}cc.tuat.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The rapid effects of steroid hormones on Ca2+ signals have been examined in bovine adrenocortical cells. Among the steroid molecules tested, only corticosterone rapidly stimulated Ca2+ signals upon addition of ACTH, although corticosterone alone did not induce Ca2+ signals. Corticosterone also enhanced steroidogenesis induced by ACTH. The enhancement of ACTH-induced Ca2+ signals was also observed with membrane-impermeable corticosterone conjugated to BSA and was not inhibited by cycloheximide. In addition, corticosterone did not enhance Ca2+ signals induced by ATP or angiotensin II. These results suggest that corticosterone selectively stimulates ACTH-induced Ca2+ signals in a nongenomic way by acting on a target in the plasma membrane. Furthermore, the supernatants of cells incubated with ACTH or ATP enhanced Ca2+ signals, suggesting that steroids produced by such treatment act in an autocrine fashion. Consistent with this idea, these effects were inhibited by inhibitors of steroidogenesis (aminoglutethimide or metyrapone). These results show that steroid molecules synthesized in adrenocortical cells facilitate ACTH-induced Ca2+ signals. Taken together, corticosterone secreted from adrenocortical cells activates ACTH-induced Ca2+ signals and steroidogenesis by nongenomic means.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
STEROID HORMONES ACT through acute and nongenomic means as well as by effects on gene transcription (1, 2). The latter effects are mediated by binding of the steroid hormone to classical steroid hormone receptors. Progesterone and corticosterone exemplify steroid hormones that also act through nongenomic means. Progesterone blocks voltage-gated and calcium-activated K+ channels on T lymphocytes (3), stimulates protein tyrosine phosphorylation by the progesterone receptor on the cell surface of human sperm (4), and induces the maturation of Xenopus oocytes by inducing association of the classical progesterone receptor with phosphatidylinositol 3-kinase (5). Corticosterone increases Na+/H+ exchange in vascular smooth muscle cells by activating protein kinase C (PKC) (6) and inhibits nicotine-induced calcium influx in PC12 cells through the pertussis toxin-sensitive, G protein-PKC pathway (7).

Treatment of adrenocortical cells with ACTH stimulates steroidogenesis in these cells, leading to the production of steroids such as progesterone and corticosterone. ACTH-induced steroidogenesis in these cells is mediated by Ca2+ (8, 9, 10, 11), cAMP (12, 13) and arachidonic acid metabolites (14, 15). An increase in the intracellular calcium concentration ([Ca2+]i) is a good indicator of steroidogenesis, because Ca2+ enhances the availability of cholesterol to cytochrome P450scc, which is the rate-limiting step in overall steroidogenesis (16, 17, 18, 19). In adrenocortical cells, ACTH induces oscillatory changes or sustained increases in [Ca2+]i that can be observed by fluorescence microscopy (20, 21). The percentage of cells that show these changes in [Ca2+]i upon stimulation increases with the concentration of ACTH (11, 20).

It has been unclear whether steroid molecules modulate signal transduction by nongenomic means in steroid-producing adrenocortical cells, although the binding of corticosterone to the plasma membrane has been reported (22). Here we report that corticosterone released from adrenocortical fasciculata-reticularis cells acts at the plasma membrane and selectively enhances ACTH-induced Ca2+ signals and steroidogenesis by nongenomic means.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Isolation and primary culture of bovine adrenocortical cells
Fresh bovine adrenal glands were obtained from a local slaughterhouse, and adrenocortical fasciculata-reticularis cells were isolated aseptically by use of collagenase as described previously (20). Briefly, adrenal cortex was minced and incubated with collagenase (0.1%) in Krebs-Ringer bicarbonate buffer [125.0 mM NaCl, 6.0 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 1.2 mM CaCl2, 25.3 mM NaHCO3, 2 mg/ml glucose, 3 mg/ml BSA, and 0.005% DNase I (pH 7.4)] for 1 h at 37 C under a gas phase of 95% O2-5% CO2 mixture. The isolated cells were cultured in Ham’s F-10 medium supplemented with 5% fetal calf serum, 10% newborn calf serum, and 2.5% horse serum in glass-bottom culture dishes coated with collagen in a CO2 incubator (humidified atmosphere of 5% CO2 in air) at 37 C. Cells cultured for 4 d were used for the experiments.

Measurement of changes in [Ca2+]i
One hour before the experiments, the cells were washed with physiological saline containing 120.0 mM NaCl, 4.0 mM KCl, 1.0 mM NaH2PO4, 0.5 mM MgSO4, 1.25 mM CaCl2, 4.0 mM NaHCO3, 1 mg/ml glucose, 1 mg/ml BSA, and 10.0 mM HEPES (pH 7.4). To load cells with Calcium Green-1, a fluorescent Ca2+ indicator, cells were incubated with 4 µM Calcium Green-1 acetoxymethyl ester in the presence of 0.05% cremophor EL for 30 min at 37 C (11, 20). The concentration of Calcium Green-1 acetoxymethyl ester was determined spectrophotometrically based on the knowledge that {epsilon} 302 nm = 17 mM-1 cm-1. Then the cells were washed three times with saline. The glass-bottom culture dishes were placed on the stage of an inverted epifluorescence microscopy (IX-70, Olympus, Tokyo, Japan) equipped with a x20 objective (Uapo x20/340, Olympus). Changes in [Ca2+]i were measured by monitoring changes in the fluorescence intensity of Calcium Green-1. Fluorescence was elicited by illumination with a 75-watt xenon lamp through a 20-nm bandpass filter centered at 480 nm. Fluorescence at more than 505 nm was monitored with a cooled CCD camera (PentaMAX 1317K 5MHz, Princeton Instruments, Trenton, NJ). A series of image frames was acquired at intervals of 3 sec, binning pixels 4 x 4, under computer control. The exposure time for each frame was 1 sec. During the following 2 sec, we blocked the light with a mechanical shutter to avoid potential cellular damage. The intensity of illumination was reduced to 12% with a neutral density filter to avoid photodynamic injury of the cells. The readout was digitalized to 12 bits and analyzed with image-processing software (MetaMorph, Universal Imaging Corp., Downingtown, PA).

Measurement of ACTH-induced steroidogenesis
Adrenocortical cells grown to confluence on a 24-well culture plate (Sumitomo Bakelite, Tokyo, Japan) were incubated in fresh medium containing the inhibitors of pregnenolone metabolism, 2 µM trilostane and 20 µM SU-10603 (supplied by C. R. Jefcoate), and ACTH. After incubation of the cells for 30 min with various concentrations of corticosterone or cortisol under 5% CO2 at 37 C, accumulated pregnenolone was extracted with hexane and measured by specific RIA, as described previously (23). Cell morphology did not change during the 30-min incubation with the inhibitor.

ACTH-induced synthesis of corticosterone or cortisol was measured after incubation of cells in the absence of trilostane and SU-10603. Corticosterone and cortisol were extracted with chloroform and separated by a silicagel column (Cosmosil 5SL, Nacalai Tesque, Kyoto, Japan; 4-mm diameter x 150 mm) with the mobile phase of hexane, which contained 0.1% acetic acid and 2-propanol of 8%, at flow rate of 0.7 ml/min. Corticosterone was eluted 37 min after injection. A fraction of elute at 35–38 min was collected, and its corticosterone content was determined by specific RIA. The amount of cortisol was estimated from the peak areas of UV absorption at 254 nm (24).

Materials
The materials were purchased from the following sources: 4-pregnen-11ß,21-diol-3,20-dione 3-CMO-BSA (corticosterone-BSA; corticosterone/BSA, 20:1) from Steraloids, Inc. (Newport, RI); ACTH1–24 from Daiichi Seiyaku Co. (Osaka, Japan); angiotensin II from Peptide Institute, Inc. (Osaka, Japan); Calcium Green-1 acetoxymethyl ester from Molecular Probes, Inc. (Eugene, OR); metyrapone from Aldrich (Milwaukee, WI); aldosterone, 18-hydroxycorticosterone, and 18-hydroxydeoxycorticosterone from Makor Chemicals Ltd. (Jerusalem, Israel); cortisone from Nacalai Tesque (Kyoto, Japan); and aminoglutethimide, cycloheximide, and other steroids from Sigma-Aldrich Corp. (St. Louis, MO). All other chemicals were of the highest purity commercially available. Each steroid was dissolved in ethanol or propylene glycol. During measurements of Ca2+ signals, the concentrations of both solvents in saline were kept at 0.005%.

A stock solution of corticosterone-BSA was made by dissolving the powder in Tris buffer (pH 7.4) at 0.03–0.1 mg/ml (load). An aliquot (100 µl) of the solution was applied to a Biospin-6 column with a 6-kDa cut off (Bio-Rad Laboratories, Hercules, CA) and subjected to centrifugation at 1000 x g for 4 min. More than 80% of BSA molecule in solution applied to the spin column was recovered in the filtrate.

Data analysis
We averaged the data produced using cells prepared from at least three independent samples of adrenal cortex. The results were expressed as the mean ± SEM and were analyzed by ANOVA followed by Bonferroni correction, or analyzed by unpaired t test. The difference was considered statistically significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of steroid molecules on Ca2+ signals in adrenocortical cells
We examined the effects of steroids on changes in [Ca2+]i upon stimulation with a physiological stimulus (Ca2+ signals) using real-time fluorescence microscopy. Ca2+ signals were monitored in individual cells, allowing us to calculate the percentage of cells that responded to the stimulus. Ca2+ signals evoked by ACTH were observed as an elevation in [Ca2+]i (Fig. 1AGo) or a perturbation of Ca2+ oscillations (Fig. 1BGo). The percentage of the cells that responded to ACTH increased from 10 ± 5% (1 pM ACTH) to 94 ± 4% (100 pM ACTH; Fig. 1CGo). At 10 pM, ACTH induced Ca2+ signals in 37 ± 4% of cells without the addition of steroid molecules. ATP (10 nM) and angiotensin II (10 pM) induced Ca2+ signals in 40 ± 1% and 24 ± 6% of cells, respectively. More than 90% of cells showed Ca2+ signals upon the addition of 100 nM ATP or 100 pM angiotensin II.



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  		FIG. 1. The time course of changes in fluorescence intensity of Calcium Green-1 in a single cell in response to ACTH. The vertical scale represents the ratio of fluorescence intensity change vs. the initial fluorescence intensity [(F/Fo - 1) x 100]. At time zero, 10 pM ACTH was added. Calcium signals appeared as an increase in [Ca2+]i (A) or as a perturbation of Ca2+ oscillations (B). C, Measurement of the fraction of cells responding to ACTH relative to ACTH concentration. Values represent the mean ± SEM. *, P < 0.05 vs. 1 pM ACTH.

 
To investigate the rapid effects of steroids on ACTH-induced Ca2+ signals, cells were incubated with steroids (100 nM) for 3 min before the addition of ACTH (10 pM). Among the steroids tested, only corticosterone enhanced ACTH-induced Ca2+ signals (by 28 ± 5%; Fig. 2AGo). In contrast, corticosterone did not stimulate Ca2+ signals induced by ATP or angiotensin II (Fig. 2BGo) and had no effect on [Ca2+]i in the absence of other stimuli (data not shown). These results indicate that corticosterone and ACTH act synergistically to enhance the mobilization of Ca2+ in adrenocortical cells. Steroid-induced increases in the fraction of cells responding to the various stimuli were analyzed, because the percentages of cells responding to stimuli varied significantly depending upon the preparation of cells. The percentage of cells that responded to treatment with 10 pM ACTH generally ranged from 30–50%.



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  		FIG. 2. Effects of steroids on Ca2+ signals. A, Effects of steroids on ACTH-induced Ca2+ signals. The percentages of cells that showed Ca2+ signals upon stimulation with ACTH (10 pM) were measured in the presence or absence of steroid. The concentrations of steroids were 100 nM if not indicated. The vertical axis represents the increase in the percentage of cells responding to ACTH (10 pM; the percentage in the presence of steroid or solvent for steroids - that in the absence of solvent for steroids). Control samples were measured in the presence of 0.005% ethanol or 0.005% propylene glycol. Values represent the mean ± SEM. *, P < 0.05 vs. control. B, Effects of corticosterone on Ca2+ signals induced by ACTH (10 pM), ATP (10 nM), or angiotensin II (10 pM). The vertical axis represents the increase in the percentage of cells responding to the indicated stimulus (the percentage in the presence of steroid or 0.005% ethanol - that in the absence of ethanol). Control samples were measured in the presence of 0.005% ethanol. Values represent the mean ± SEM. *, P < 0.05.

 
In all cases tested, stimulus-induced changes in [Ca2+]i consisted of the rapid increase within 100 sec and the following sustained changes. We observed three types of sustained changes, i.e. oscillation, sustained increase, and oscillation superimposed on sustained increase. When cells were stimulated with 10 pM ACTH, we observed oscillation in 30%, sustained increase in 10%, and oscillation superimposed on sustained increase in 60% of cells that responded to ACTH. These percentages were not affected by any of the steroids tested, although corticosterone increased the percentage of cells that showed the rapid increase in [Ca2+]i. Therefore, we analyzed Ca2+ signals by calculating the percentage of cells that showed the rapid increase in [Ca2+]i upon stimulation. It should be noted that corticosterone affected neither the rapid increase nor the following sustained changes in [Ca2+]i when cells were stimulated with ATP or angiotensin II.

Effects of corticosterone on steroidogenesis
As corticosterone significantly enhanced ACTH-induced Ca2+ signals, we examined whether corticosterone also enhanced steroidogenesis induced by ACTH. The effects on steroidogenesis were assayed by measuring the formation of pregnenolone, the rate-limiting step in overall steroidogenesis (13, 25). In Fig. 3Go we show that in the presence of ACTH (1–100 pM), corticosterone significantly enhanced pregnenolone synthesis. On the other hand, cortisol, which was used as a control, did not stimulate pregnenolone synthesis (data not shown). Similar to the ACTH specificity of the effects on the Ca2+ signal, corticosterone did not increase pregnenolone synthesis that was induced by ATP (data not shown). In the absence of exogenous corticosterone and inhibitors, the concentration of corticosterone increased to 54 ± 4 nM under the experimental condition (corresponding to 90 ± 7 pmol/mg protein) after stimulation of cells with 1 pM ACTH for 30 min.



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  		FIG. 3. Effect of corticosterone (100 nM, 30 min) on the dose-dependent stimulation of pregnenolone synthesis by ACTH. The cultured cells were incubated with or without corticosterone in the presence of trilostane and SU-10603 to generate pregnenolone. Values represent the mean ± SEM. *, P < 0.05.

 
Nongenomic actions of corticosterone on ACTH-induced Ca2+ signals via the plasma membrane
As corticosterone acts on the ACTH-mediated pathway within 3 min of its application to cells, its effects probably do not involve de novo protein synthesis. To confirm this hypothesis, we examined the effect of cycloheximide on corticosterone action. Treatment of cells with cycloheximide at 40 µM for 30 min did not affect the enhancement of ACTH-induced Ca2+ signals by corticosterone (Fig. 4Go), although treatment with cycloheximide resulted in significant suppression of de novo synthesis of steroidogenic acute regulatory protein upon stimulation with ACTH (data not shown). This result indicates that the action of corticosterone on ACTH-induced Ca2+ signals does not rely on de novo protein synthesis. As cycloheximide suppresses steroidogenesis upon stimulation with ACTH, cycloheximide might also suppress ACTH-induced Ca2+ signals through inhibition of corticosterone production. When cells were incubated with cycloheximide for 30 min in the absence of stimuli, however, the possibility was excluded, because the concentration of accumulated corticosterone in cell cultures was determined to be less than 10 nM after incubation of cells for 30 min without stimulation.



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  		FIG. 4. Nongenomic effects of corticosterone on ACTH-induced calcium signals. Cells were incubated with corticosterone, cycloheximide (CHX), CHX plus corticosterone, RU486, RU486 plus corticosterone, corticosterone-BSA, corticosterone-BSA plus RU486, or the filtrate after application of free corticosterone (100 nM) to a Biospin-6 column, and then stimulated with ACTH (10 pM). Corticosterone (100 nM), RU486 (1 µM), and corticosterone-BSA (5 nM) were added to cells 3 min before the addition of ACTH. The vertical axis represents the increase in the percentage of cells responding to ACTH (the percentage in the presence of indicated agents or 0.005% ethanol - that in the absence of ethanol). Control samples were measured in the presence of 0.005% ethanol. Values represent the mean ± SEM. *, P < 0.05 vs. control.

 
The nongenomic effects of steroid hormones can be dependent upon binding to sites in or on the plasma membrane (4, 26, 27, 28). To determine whether binding sites for corticosterone exist in the plasma membrane, we treated cells with membrane-impermeable conjugates of corticosterone and BSA. Corticosterone-BSA conjugates were added for 3 min at a concentration of 5 nM. The addition of corticosterone-BSA conjugates to the cells significantly enhanced ACTH-induced Ca2+ signals (Fig. 4Go). We also treated cell with RU486 (1 µM), an antagonist of glucocorticoid receptor. We found that RU486 did not affect the corticosterone action on ACTH-induced Ca2+ signals. These results suggest that corticosterone enhances ACTH-induced Ca2+ signals in a nongenomic manner by acting upon putative binding sites on the plasma membrane, and not by modulating protein synthesis.

Steroid molecules produced in adrenocortical cells enhance ACTH-induced Ca2+ signals
As adrenocortical cells produce corticosterone, the steroid might enhance ACTH-induced Ca2+ signals in an autocrine fashion. To examine this possibility, we first incubated cells with 1 pM ACTH or 10 nM ATP for 30 min at 37 C to stimulate corticosterone synthesis. We then measured the effects of the supernatant from the above cells on ACTH (10 pM)-induced Ca2+ signal. Pretreatment of cells for 3 min with the supernatant from cells previously incubated with ACTH (1 pM) or ATP (10 nM) enhanced the ACTH (10 pM)-induced Ca2+ signal by 44 ± 8% or 26 ± 5%, respectively (Fig. 5Go). We next incubated cells with 1 pM ACTH or 10 nM ATP for 30 min in the presence of 200 µM aminoglutethimide (AG) or 10 µM metyrapone (MP). Under these conditions, we confirmed that AG decreased pregnenolone formation induced by 1 pM ACTH to 10%, and that MP decreased the activity of 11ß-hydroxylase to less than 1% without significantly decreasing P450scc activity. Both inhibitors also completely blocked the effects of the supernatants on ACTH-induced Ca2+ signals, indicating that steroid molecules released from adrenocortical cells enhance ACTH-induced Ca2+ signals. Taken together, these results suggest that corticosterone acts in an autocrine fashion. It should be noted that AG and MP did not affect the sustained changes in [Ca2+]i, although they decreased the percentages of cells that responded to ACTH.



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  		FIG. 5. Effects of supernatants from cells incubated with ACTH, ATP, or inhibitors of steroidogenesis on ACTH-induced Ca2+ signals. Cells were incubated with ACTH (1 pM), ATP (10 nM), or physiological saline without the stimulus. Inhibition of steroidogenesis was performed with AG (200 µM) or MP (10 µM). After replacement of the medium with the supernatants from cells incubated with the indicated agents, ACTH-induced Ca2+ signals were measured. The vertical axis represents the increase in the percentage of cells responding to ACTH (10 pM) by replacing the medium with the supernatants. Values represent the mean ± SEM. *, P < 0.05 vs. physiological saline.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study we found that 1) corticosterone specifically enhances calcium signals and pregnenolone synthesis in adrenocortical cells in response to treatment with ACTH; 2) the corticosterone effect on calcium signals appears within minutes and is not blocked by an inhibitor of protein synthesis; 3) the putative binding site of corticosterone exists in or on the plasma membrane; and 4) the effect of corticosterone is specific to ACTH-mediated signals.

Upon stimulation with ACTH at concentrations of up to 100 pM, the percentage of cells that showed increases in [Ca2+]i correlated with steroidogenesis (11, 20). Ca2+ channel blockade also suppressed ACTH-induced steroidogenesis (8, 10, 20, 29), indicating that Ca2+ influx is important for ACTH-induced steroidogenesis in bovine adrenocortical cells. Taken together, these results suggest the hypothesis that Ca2+ is a second messenger for ACTH. The present observation that corticosterone simultaneously activated calcium signals and steroidogenesis (pregnenolone synthesis) is consistent with the above hypothesis. Further, in addition to ACTH, ATP (30) and angiotensin II (31, 32, 33) require the increase in [Ca2+]i to stimulate steroidogenesis. This is probably because Ca2+ increases the availability of cholesterol to cytochrome P450scc (16, 17, 18, 19, 34), which is the rate-limiting step in overall steroidogenesis. In addition to the increase in the availability of cholesterol, the elevation of [Ca2+]i can activate enzymes involved in the synthesis of NADPH, higher levels of which can also enhance steroidogenesis (35). cAMP and arachidonic acid metabolites might also contribute to the observed enhancement of steroidogenesis by corticosterone, as these two molecules also act as second messengers in adrenocortical cells (12, 13, 14, 15).

The rapid enhancement of calcium signals by corticosterone suggests that the effects are not due to changes in protein synthesis mediated by the classical steroid receptor pathway. The protein synthesis inhibitor cycloheximide did not affect corticosterone action on ACTH-induced Ca2+ signals, further supporting the nongenomic origin of its effects. A putative membrane receptor has been suggested to mediate the nongenomic effects of corticosterone (7, 36). Consistent with this idea, a membrane-impermeable corticosterone-BSA conjugate also induced activation of calcium signals upon addition of ACTH. The presence of corticosterone-binding sites in the plasma membrane of bovine adrenocortical cells was strongly suggested by radioactive binding studies using [3H]corticosterone (22). In the latter experiments, it was demonstrated that corticosterone bound to a specific class of proteins with a binding constant of 77 nM. These results suggest that corticosterone acts upon binding sites in the plasma membrane and nongenomically activates ACTH-induced Ca2+ signals, resulting in the stimulation of steroidogenesis. Recently, it was reported that estrogen receptors associated with the plasma membrane are possibly the same as nuclear estrogen receptors and activate G proteins (37). In addition to estrogen receptors, nuclear glucocorticoid receptors are reported to associate with the plasma membrane in neurons (38). Therefore, nuclear glucocorticoid receptors might be involved in the corticosterone action on ACTH-induced Ca2+ signals.

In addition to their interaction with specific receptors, steroid effects may be induced by nonspecific membrane interaction (39). However, the effects of corticosterone observed here were ACTH dependent and were not induced by other steroid molecules. Therefore, it is conceivable that the effects were due to the specific interaction of corticosterone with a membrane receptor. Furthermore, our results suggest that the effects of corticosterone might be physiologically relevant, as the levels used in this study (100 nM), although slightly higher than those present in human serum, might be similar to levels present in steroid-producing adrenocortical cells. It should be noted that cortisol does not affect ACTH-induced Ca2+ signals and steroidogenesis, although it is principal glucocorticoid produced by bovine adrenal glands.

In summary, the main conclusion of the current study is that corticosterone probably activates both ACTH-induced calcium signals and steroidogenesis by binding a receptor in the plasma membrane. This is consistent with previous findings indicating that corticosterone-binding proteins exist in the plasma membrane (22). As corticosterone is produced in fasciculata cells and glomerulosa cells, this molecule may act physiologically as an autocrine signal molecule and/or a paracrine signal molecule to enhance steroidogenesis. The present results represent significant progress in understanding the nongenomic action of corticosterone on steroidogenesis, although the proteins and the mechanisms involved in this action remain to be identified.


   Acknowledgments
 
We thank Prof. M. Kawamura and Dr. H. Nishi (Department of Pharmacology, Jikei University School of Medicine) for their helpful discussions. We also thank Ms. A. Matsuyama for her technical assistance.


   Footnotes
 
This work was supported by grants from the Ministry of Education, Science, and Culture, Japan.

Abbreviations: AG, Aminoglutethimide; [Ca2+]i, intracellular calcium concentration; MP, metyrapone; PKC, protein kinase C.

Received October 28, 2002.

Accepted for publication April 30, 2003.


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 Results
 Discussion
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C. E. Mohn, J. Fernandez-Solari, A. De Laurentiis, J. P. Prestifilippo, C. de la Cal, R. Funk, S. R. Bornstein, S. M. McCann, and V. Rettori
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