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Endocrinology Vol. 140, No. 11 5154-5164
Copyright © 1999 by The Endocrine Society

ARTICLES

The in Vivo Effects of Adrenocorticotropin and Sodium Restriction on the Formation of Different Species of Steroidogenic Acute Regulatory Protein in Rat Adrenal1

Jean-Guy Lehoux, Dale Buchanan Hales, Alain Fleury, Normand Brière, Denis Martel and Lyne Ducharme

Department of Biochemistry (J.-G.L., A.F., N.B., D.M., L.D.), Faculty of Medicine, University of Sherbrooke, Sherbrooke, Québec, Canada J1H 5N4; and Department of Physiology and Biophysics (D.B.H.), University of Illinois at Chicago, Chicago, Illinois 60612-7342

Address all correspondence and requests for reprints to: Jean-Guy LeHoux, Department of Biochemistry, Faculty of Medicine, University of Sherbrooke, Sherbrooke, Québec, Canada J1H 5N4. E-mail: jlehou01{at}courrier.usherb.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have studied the in vivo expression of steroidogenic acute regulatory protein (StAR) in adrenals of control, ACTH-treated, and Na+-restricted rats. Indirect immunofluorescence by microscopy revealed the presence of StAR in the zonae glomerulosa (ZG) and fasciculata-reticularis (ZFR). An increased signal was observed in the ZG and zona fasciculata, 5 h after ACTH injection; a few cells of the medulla were also positive. Immunogold electron microscopy showed that StAR was mainly located over mitochondria (MT). By immunoblotting, a major 29-kDa and other minor StAR bands migrating between 30 and 39 kDa were increased 5 h after ACTH treatment but remained unchanged after 1 h. By two-dimensional-PAGE, four StAR species were revealed in homogenates of control ZG, and their intensity was increased 5 h after ACTH treatment but not after 1 h. Also, additional acidic species were seen 5 h after treatment. Other bands with basic isoelectric point were revealed between 29 and 37 kDa. Analyses on whole gland MT and supernatant (SN) revealed four bands in the control SN and five in ACTH SN; the intensity of one band was increased, and that of another one was decreased, in SN of treated rats. ACTH treatment resulted in the localization of many low-isoelectric point StARs in MT. After two-dimensional-PAGE, differences were found in the mobility of some StAR species in the ZG between controls and Na+-restricted rats. In MT, four bands were revealed in the ZG preparations of Na+-restricted and two bands in controls. Four bands were revealed in the ZG SNs of control and Na+-restricted rats; an additional band was observed only in the SN of treated animals, whereas the intensity of another band decreased. Na+ restriction did not affect StAR in the ZFR. In conclusion, StAR was present in the rat adrenal cortex ZG and ZFR and was mainly located in MT. StAR expression was inducible in the ZG and the ZF by ACTH, resulting in the formation of many StAR acidic species; interestingly, such changes were detectable 5 h, but not 1 h, after ACTH administration, suggesting that steroidogenesis stimulation by StAR might occur mainly outside MT. Although less spectacular than for ACTH, Na+ restriction also affected StAR expression in the ZG but not in the ZFR, by increasing two mitochondrial and one SN species, implying that StAR is involved in the mechanism of action of Na+ restriction in promoting aldosterone formation. These results suggest that differential processing and/or changes in phosphorylation may occur in vivo upon ACTH treatment and Na+ restriction. We hypothesize that modification of a relatively small quantity of StAR, mainly located outside MT, is necessary to increase adrenal steroidogenesis challenged either by ACTH or Na+ restriction.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CHOLESTEROL IS THE precursor of aldosterone in the adrenal cortex zona glomerulosa (ZG) and of glucocorticoids in the zonae fasciculata and reticularis (ZFR) (1). The first and rate-limiting step of steroid biosynthesis is the transformation of cholesterol to pregnenolone by the cholesterol side-chain cleavage system cytochrome P450 (P450scc), localized on the matrix side of the inner mitochondrial membrane. Under acute stimulation, the quantity of pregnenolone formed (2, 3) is attributable to the availability of cholesterol to P450scc and not to the activity of this enzymatic system (4). Indeed, it was shown that the inhibitor of protein synthesis cycloheximide inhibited the transfer of cholesterol across the intermembrane aqueous space in mitochondria (MT) and concomitantly blocked steroidogenesis (5, 6).

Clark et al. (4) reported that steroidogenic acute regulatory protein (StAR) plays a crucial role in steroidogenesis by enhancing the delivery of cholesterol to P450scc. In a disease named lipoid congenital adrenal hyperplasia, there is accumulation of cholesterol in cytoplasmic lipid droplets, and gonadal and adrenal steroidogenesis is impaired; mutations in the StAR gene were shown to be responsible for defective steroidogenesis (7, 8, 9, 10, 11, 12). Furthermore, impaired steroidogenesis and lipid accumulation in steroidogenic tissues were obtained in StAR gene nullizygous mice produced by homologous recombination with identical phenotype (13). These biochemical and genetic data have been interpreted as indicating that StAR is a key molecule in controlling cholesterol transfer across the mitochondrial membrane and, consequently, in controlling the transformation of cholesterol to pregnenolone. It has been reported that StAR has sterol transfer activity, which may reflect an ability to enhance desorption of cholesterol from sterol-rich donor membranes (14).

Arakane et al. (15) recently reported that deletion of up to 62 residues from the N terminus of StAR did not affect its steroidogenic activity, although its importation into MT was prevented. In agreement, we found (16) that in vivo administration of ACTH to rats resulted in a rapid increase in adrenal steroidogenesis, without affecting the quantity of adrenal homogenate or mitochondrial StAR; in fact, an increase in StAR was observed only 3 h after ACTH administration in both homogenate and MT.

Two main factors are regulating steroidogenesis in the adrenal cortex, ACTH and angiotensin II (AII), both regulating steroid formation at the site of transformation of cholesterol to pregnenolone (17). The regulation of steroidogenesis by ACTH is mediated by cAMP (1), but administration of this hormone was also shown to produce, within a few minutes, increases in the expression of protooncogenes of the Jun/Fos family, suggesting a role for these factors in acute stimulation by ACTH (16). The effect of sodium restriction is mediated by AII (18) through its binding to AII receptors. Although the signaling pathway used by AII differs from that of ACTH, AII was able to induce StAR expression in adrenocortical cells, suggesting that StAR might also be part of the mechanism of action of AII (19).

In vitro studies on steroidogenic cells in culture, using 35S-methionine or 32P-orthophosphate, revealed that ACTH and AII affected the labeling of many proteins (20, 21) now identified as StAR species. Moreover, using COS-1 cells to express human StAR, a mutation of a potential site for protein kinase A-mediated phosphorylation at serine 195 to alanine reduced 32P incorporation from labeled ATP into StAR. The capacity of that mutant to induce pregnenolone production was reduced by 50% (22), indicating the importance of phosphorylation as part of the mechanism of action of this protein to control steroidogenesis.

The objective of this study was to characterize the pattern of StAR expression in rat adrenal ZG and ZFR, comparing the acute effects of ACTH administration in vivo with those of AII, induced by sodium restriction. To detect differences provoked by these treatments, proteins were separated by electrophoresis or by electrofocusing/electrophoresis before immunoblotting. Western blotting analysis, using an anti-StAR antibody directed against the mature part of the mouse StAR molecule (23), showed an increase in StAR species of different molecular weights in both ZG and ZFR, 3 h and 5 h after ACTH treatment but not after 1 h. Many acidic StAR species were also revealed 5 h after treatment.2 Although quantitatively less spectacular than for ACTH, sodium restriction induced the formation of StAR species of different isoelectric points in the ZG but not in the ZFR. Our results suggest that differential processing and/or changes in phosphorylation may occur in vivo upon ACTH treatment and sodium restriction.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and treatments
Two-month-old male Long Evans rats were purchased from Charles River Laboratories, Inc. (St. Constant, Québec, Canada) and were kept on Purina rat chow and tap water ad libitum. Each rat received a single im injection of ACTH in the gluteal region (1 U/100 g BW of Synacthen Depot, Ciba Pharmaceuticals, Division of Ciba-Geigy Canada Ltd., Mississauga, Ontario, Canada). Animals were killed (in accordance with the ethical standards of the institutional review committee) at different times after the first injection, as specified later in Results. Other groups of rats were kept on a sodium-deficient diet, (<0.01 mEq Na+/g; ICN Biochemicals, Inc., Cleveland, OH) with demineralized water to drink, for 11 days, as previously described (17). Rats were killed by decapitation, and blood was collected. The adrenals were removed, freed of fat, and used as detailed below. The ZG was separated from the ZFR plus the medulla by the method of Giroud et al. (25).

Sample preparation
Tissues were homogenized in 50 mM Tris (pH 7.4), 0.25 M sucrose, 5 mM EDTA, 1 mM phenylmethylsulfonylfluoride, 0.1 mM leupeptin, 30 mM iodoacetamide, 0.125 µM aprotinin, using a Potter-Elvehjem homogenizer with a loosely fitting Teflon pestle. A portion of the homogenate was centrifuged 10 min at 900 x g, and the supernatant (SN) was then centrifuged 15 min at 9,500 x g. The last SN was kept, and mitochondrial pellet was resuspended in homogenization buffer. Then, cholic acid (1% wt/vol) and SDS (0.1% wt/vol) were added to all preparations. These were frozen in liquid nitrogen, freeze-thawed twice, and solubilized in Laemmli sample buffer (26). They were passed through a 26-gauge needle, then boiled for 5 min, and finally centrifuged at 12,000 x g for 2 min.

One-dimensional (1D) gel electrophoresis
Soluble proteins were electrophoresed on 12% polyacrylamide gel (SDS-PAGE) and analyzed by immunoblotting, as previously described (27, 28), using a rabbit polyclonal antimouse StAR protein antibody; this Ig was raised against a GST-fusion protein from a mouse StAR complementary DNA (cDNA) from bp208–1467 fragment (23). Immunoreactive proteins were detected using enhanced chemiluminescence light emitting reagents (Amersham International plc., Little Chalfont, Buckinghamshire, UK). Autoradiograms were observed by exposing the blots to Kodak X-Omat RP films (Amersham Pharmacia Biotech, Baie d’Urfé, Québec, Canada). The intensity of bands on the films was determined using an LKB 2222–020 Ultroscan XL scanning laser densitometer (Pharmacia Canada Ltd., Baie d’Urfé, Québec, Canada).

Two-dimensional (2D) gel electrophoresis
Gel rods (110 mm x 4 mm) were made with 8 M urea, 4.5% polyacrylamide, 2% Triton X-100, and 2% ampholines (0.67% pH 3–10 and 1.33% pH 5–7). Prefocusing (0.66 mA per gel rod) was performed during 1 h with a 500-V maximum limit in NaOH 0.1 N (catholyte) and 0.06% phosphoric acid (anolyte). Soluble proteins from SN, homogenate, or mitochondrial preparations were diluted with 2 vol sample dilution buffer, which contained 9.5 M urea, 10% Triton X-100, 2% ampholines, 5% mercaptoethanol (vol/vol). Samples were loaded and overlayed with 5 M urea, 2% Triton X-100, and 2% ampholines. Constant 400 V was applied for 9000–10000 volt hours. Gel rods were equilibrated 2 x 20 min in a reducing buffer [62.5 mM Tris (pH 6.8), 5% ß-mercaptoethanol, 3% SDS, 0.01% bromophenol blue]. Reduced rods may be stored at -80 C until use. Thawed rods were fixed with 1% agarose in upper buffer [125 mM Tris (pH 6.8), 0.1% SDS]. SDS-PAGE and immunoblotting were performed as described above. Isoelectric points were determined using 2D SDS-PAGE standards no. 161-0320 from Bio-Rad Laboratories, Inc. (Mississauga, Ontario, Canada). In addition, in some experiments, standards and adrenal preparations were mixed and run on the same gel. After transferring onto membranes, standards were stained, and StAR was detected by enhanced chemiluminescence, as described for 1D gels.

Immunolocalization
Immunofluorescence. For the localization of StAR, adrenal glands were excised from three different animals in each experimental group. One gland from each animal was fixed in buffered neutral formalin solution for 24 h. The fixed adrenals were dehydrated in graded alcohols, cleared in toluene, and embedded in paraffin. Five to seven 5-µm-thick sections were prepared according to the usual histologic procedure. Sections were deparaffinized, hydrated to water, and treated with NH4Cl in 50 mM PBS (20 min) to block aldehydes. After two washes, tissue sections were incubated for 2 h at room temperature with the antimouse StAR antibody (diluted 1:100 in PBS containing 1% BSA) and then washed twice. The slides were then incubated for 30 min with a fluorescein-conjugated goat antirabbit IgG (Roche Molecular Biochemicals, Mannheim, Germany) diluted 1:50, washed in PBS for 5 min, and then mounted in glycerol-PBS (9:1) containing 0.1% phenylenediamine (29). Control sections were incubated with the second antibody only. Paraffin-embedded sections were studied using a Reichert Polyvar 2 microscope equipped for epifluorescence.

Immunocytochemistry. The other gland from each animal was cut into pieces, which were fixed in ice-cold 3% paraformaldehyde, 1% glutaraldehyde for 3 h at 4 C and then embedded in LR White resin (Electron Microscopy Sciences, Fort Washington PA). Four to five thin sections (about 750 Å) from each gland were deposited on two to three coated grids and were treated for 10 min with Blotto 10%. The sections were then incubated overnight at 4 C with the antimouse StAR antibody, diluted 1:20, and washed three times in PBS and PBS-BSA. They were next incubated for 30 min with 1:10 gold-conjugated (30) goat antirabbit IgG (10 nm gold particles; Cedarlane Laboratories, Hornsby, Ontario, Canada), washed in bidistilled water, and stained with uranyl acetate and lead citrate. Tissues were then examined by electron microscopy. For the quantitative analysis, six different areas were randomly selected in the ZG and in the zona fasciculata of the thin plastic adrenal sections. This was done for each one of the three controls and the three ACTH-treated animals of each of the following groups: two groups of three rats were administered ACTH and killed 1 h and 5 h after treatment. Thus, for each animal, the number of gold particles was determined in 60 MT, 6 cytoplasmic, and 6 nuclear areas using an electron microscope (Jeol JEM-100 CX) equipped with a CCD camera system (Talktronics, Lake Forest, CA). After, the number of gold particles present in each 1 µm2 of the above 3-cell compartments was determined.

Statistical analysis
Differences between experimental groups were analyzed by ANOVA followed by Dunnett’s test, using the SigmaStat program for Windows (Jandel Corporation, San Rafael, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Immunolocalization
The antimouse StAR antibody labeled with fluorescein was used to detect and visualize the zonal distribution of StAR on paraffin sections of the rat adrenal. The protein was observed in the three zones of the adrenal cortex, although differentially distributed. In control adrenals (Fig. 1AGo), next to the negative fibroconnective capsule, the ZG appeared as a thin bright reactive band of three to four cell layers. The ZG was easily distinguished from the ZF by a narrow negative demarcation line, possibly corresponding to the intermediate zone of stem cells described by other investigators (31). However, no such clear delineation was found between the ZF and the zona reticularis. In the latter two zones (ZFR), the fluorescent signal was more diffusely and unevenly distributed, with some positive cells being intermixed with negative or weakly reactive ones.



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  		Figure 1. Indirect immunofluorescence micrograph of paraffin sections of rat adrenals at 0 (A and C) and 5 h (B and D) after ACTH stimulation. A, In control adrenals, the fluorescein-conjugated antibody revealed StAR that seems to be more concentrated in the thin ZG (arrows) but more diffusely distributed in the thick ZFR (black bar) (x 115); B, 5 h after ACTH treatment, the intensity of the fluorescent signal was mainly increased in the ZG (arrows) and the zona fasciculata (black bar) (x 115); panel C, the localization of the antibody in this control adrenal appears as bright dots in the cell cytoplasm. Nuclei (arrows) are negative. C (abbreviation), Capsule; ZG, zona glomerulosa (x 1175); D, groups of cells in the medulla are immunoreactive in this ACTH-treated animal (x 470).

 
The detection of StAR was amplified 5 h after ACTH administration (Fig. 1BGo). Indeed, the intensity of the immunofluorescent signal was perceptively increased in the ZG and ZF. The change was not apparent in the ZR, which made much easier the identification of the two contiguous zones.

As shown at higher magnification in Fig. 1CGo, the fluorescent antibody was not uniformly scattered but appeared as bright dots in the cell cytoplasm, whereas nuclei were negative, in control and in treated animals. An interesting observation, also reported by other investigators (32, 33), was the presence of StAR in groups of cells in the medulla from ACTH-treated rats (Fig. 1DGo). In controls, a few cells also appeared positive for StAR in the medullary region, although less intensely stained. Further studies are needed to characterize these cells.

To more precisely define the intracellular localization of StAR, we examined the distribution of the gold-labeled antibody by electron microscopy. Control sections incubated with the second antibody only were negatively labeled (not shown). Positive labeling with gold particles was mainly located over MT (> 98%) and much less markedly over cytoplasmic regions between MT but not over nuclei (Fig. 2Go, A and B). According to our semiquantitative evaluation, the number of colloidal gold particles seemed greater in adrenals of rats treated with ACTH than in controls. To obtain quantitative data to support our observations, the number of gold particles was determined on MT of control (Fig. 2AGo) and ACTH-treated (Fig. 2BGo) animals. Compared with control rats, in treated animals 5 h after ACTH administration, there was an increase (Table 1Go) in the number of gold particles over MT (1.7-fold, P < 0.05) in the ZG and (1.5-fold) in the ZF. Table 1Go also shows that, 1 h after treatment, there was no increases in the number of gold-particles over MT in the ZG and the ZF. The number of gold particles over cytoplasm was not significantly different in the adrenal cells of control and ACTH-treated animals (data not shown). Only a few particles, probably corresponding to background, could be observed over some of the nuclear areas analyzed. Hence, nuclei were considered as negative, in agreement with the absence of fluorescent signals in this organelle (Fig. 1CGo).



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  		Figure 2. Colloidal-gold electron microscopic studies of thin rat adrenal sections. A, In the ZG of the control glands, StAR was detected over MT. A few particles are localized in the cytoplasm, whereas the nucleus (N) is negative (x 40,000). B, The number of gold particles is increased over MT in the adrenal ZG in rats 5 h after ACTH stimulation. The detection of StAR remained unchanged in the cytoplasm and negative in the N (x 40,000). C, Higher magnification of mitochondrion (*) appearing in A and B (x 80,000).

 

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  		Table 1. Effect of ACTH administration on the number of colloidal gold particles per µm2 of mitochondria in rat adrenal

 
ACTH administration: 1D and Western blotting analysis
ACTH was administered to two groups of two rats that were killed 5 h later; two other groups served as controls. Western blotting analyzes were performed on MT and SNs obtained by differential centrifugation of homogenates of whole glands. As seen in Fig. 3Go, the anti-StAR antibody reacted against a main StAR band of 29 kDa in the SN and also in the mitochondrial preparations. Under ACTH stimulation, the intensity of five protein bands between 29–39 kDa appeared increased into MT and also that of five protein bands comprised between 29–39 kDa in SN. The 30-kDa band was seen only in mitochondrial preparations of ACTH-treated animals. Moreover, the 34-kDa protein appeared increased only in SN of ACTH-stimulated rats. Noteworthy is the fact that the major immunoreactive band was detected in the 29-kDa area; the relative intensity of the second band in importance, the 30-kDa, was not more than one tenth that of the 29-kDa band. The relative intensity of the other ACTH-sensitive StAR species was even lower than that of the 30-kDa band.



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  		Figure 3. Effects of ACTH on the level of StAR in rat whole adrenals. Two groups of three male rats were injected with ACTH and were killed 5 h after treatment; two other groups served as controls. Western blotting analyses were performed on 40 µg of mitochondrial and SN preparations, obtained by differential centrifugation, and were submitted to SDS-PAGE. Film exposure time, 2 min.

 
In another series of experiments, nine groups of four rats were treated with ACTH, and three other groups served as controls; treated animals were killed at 1 h, 3 h, and 5 h after ACTH administration. ZG and ZFR were separated, and Western analyses were performed on homogenates and MT. In the ZFR, two main StAR protein bands migrated in the area of 29–30 kDa. Figure 4Go shows that ACTH treatment increased the intensity of StAR protein bands of 29, 30, and 36 kDa in homogenates and mitochondrial preparations 3 h and 5 h after its administration. However, 1 h after ACTH treatment, a slight decrease (better seen in the lower panel with a longer film exposure) in the intensity of the 36-kDa band was observed, in both homogenate and mitochondrial preparations, to increase thereafter at 3 h and 5 h. As for the ZFR, in the ZG, the two main StAR reactive protein bands migrated in the area of 29 and 30 kDa. ACTH treatment increased the intensity of StAR protein bands of 29, 30, 33, and 36 kDa in homogenate and mitochondrial preparations 3 h and 5 h after its administration.



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  		Figure 4. A time study of the effects of ACTH on the level of StAR in rat adrenal. Western blotting analyses were performed on ZG and ZFR plus the medulla. Nine groups of four male rats were injected with ACTH and were killed at 1, 3, and 5 h after treatment, respectively; three other groups served as controls. Adrenals of each group were pooled, and analyses were performed on 50 µg of homogenate and mitochondrial proteins after SDS-PAGE. Film exposure time, 30 sec (first and third panels) and 10 min (second and fourth panels), respectively. H, Time in hours.

 
ACTH administration: 2D electrophoresis and Western blotting analyses
In the next series of experiments, we have analyzed the effects of ACTH on rat adrenal StAR species by electrofocusing/electrophoresis (2D) and immunoblotting. Analyses were performed on homogenate preparations of the ZG. Figure 5Go shows results of a representative experiment performed three times on the ZG homogenates; in control experiments, two major StAR species were revealed (pI/kDa), 5.9/29 and 5.7/29; and two minor species were found, 5.57/29 and 5.5/29. One hour after ACTH administration, there was no apparent change between preparations of control and treated animals except for the disappearance of the 5.57/29 band. Five hours after ACTH administration, however, the intensity of the above four StAR species increased. Moreover, five additional minor immunoreactive species were revealed: 6.65/31, 5.8/29, 5.4/29, 5.35/29, and 5.2/29 (arrows). Other immunoreactive bands were also revealed in the area corresponding to the origin of the electrofocusing gel, at pI of about 8.5, in the area comprised between 29 and 37 kDa and this for both control and ACTH5 h preparations. Similar results were found for the ZFR homogenate preparations (not shown).



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  		Figure 5. Effects of ACTH on the level of StAR in the ZG. Two groups of male rats were injected with ACTH and then killed 1 h and 5 h after treatment, respectively; another group served as control (CTR). Fifty micrograms of homogenate (HOM) were separated by electrofocusing and electrophoresis (2D) and transferred onto nitrocellulose membrane. Western blotting analysis was performed using a specific anti-StAR antibody. Film exposure time, 6 min. The bottom panel is a schematic that shows the mobility of the different StAR species on 2-D. Each species is expressed as pI over MW (in kDa).

 
We further have analyzed StAR by 2D on mitochondrial and SN preparations from whole adrenals. Figure 6Go shows results obtained for mitochondrial preparations of control and ACTH5 h-treated animals. In the control, two main StAR species (5.9/29 and 5.7/29) and four minor species (5.8/29, 5.57/29, 5.5/29, and 5.45/29) were revealed. Five hours after ACTH administration, all the above mentioned species seemed increased in intensity and new StAR species were revealed [5.78/30 (arrow), 5.4/29, 5.3/29, and 5.2/29] in this preparation. The 5.78/30 species was identified in MT but not in the SN. Other StAR species were revealed (8.5/29, 8.5/33, 8.5/34, and 8.5/36) in mitochondrial preparations of control, and their intensity increased after ACTH treatment. In the SN, ACTH treatment led to less apparent changes than those in MT. However, ACTH administration induced an increase in the 5.57/29 and 5.4/29 species and a diminution in the 5.4/28 species (arrow). The latter species was observed only in SN but not in MT. Furthermore, the 8.5/36 species was also revealed in SN of control and ACTH-treated animals, and its intensity was greater than in mitochondrial preparations.



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  		Figure 6. Effects of ACTH on the level of StAR in rat whole adrenals. One group of male rats was injected with ACTH and then killed 5 h after treatment; another group served as control. Analyses were performed on 30 µg of mitochondrial (MT) and SN preparations, obtained by differential centrifugation, and were submitted to electrofocusing and electrophoresis (2D). Western analysis was performed using a specific anti-StAR antibody. Film exposure time, 6 min. The bottom panel is a schematic that shows the mobility of the different StAR species on 2-D. Each species is expressed as pI over MW (in kDa).

 
In the next series of experiments, we have compared the mitochondrial StAR content of the ZG with that of the ZFR. Figure 7Go shows results of 2D analyses on mitochondrial preparations of ZG and ZFR from controls and 5 h after ACTH treatment. The 2D patterns are similar in many aspects between the ZG and ZFR preparations, although the intensity of the immunoreactive bands seemed stronger in the ZFR than in the ZG. In addition, a 5.8/29 species and two species located in the area between 5.5/29–5.4/29, observed in the ZFR, were absent in the ZG. Moreover, compared with homogenates (Fig. 5Go), two 5.78/30 and approximately 6.65/31 bands were revealed with mitochondrial preparations in the ZG and ZFR.



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  		Figure 7. Effects of ACTH on the level of StAR in rat adrenal ZG and ZFR. One group of male rats was injected with ACTH and then killed 5 h after treatment; another group served as CTR. Analyses were performed on 50 µg of mitochondrial preparations, obtained by differential centrifugation, and were submitted to electrofocusing and electrophoresis (2D). Western analysis was performed using a specific anti-StAR antibody. Film exposure time, 6 min. The bottom panel is a schematic that shows the mobility of the different StAR species on 2-D. Each species is expressed as pI over MW (in kDa).

 
The effects of sodium restriction on StAR: 1D-analysis
1D electrophoresis and Western blotting analyses were performed on homogenates of the ZG and ZFR of rats maintained on a low-sodium diet for 1 or 2 or 11 days. Compared with rats kepts on a normal diet, there were no apparent changes in the intensity of StAR immunoreactive protein bands between groups in the ZG and ZFR (results not shown).

The effects of sodium restriction on StAR 2D-analysis
We thus decided to concentrate our efforts on 2D analyses on adrenal preparations of sodium-restricted rats, for 11 days, to detect possible qualitative changes between the groups studied. Although less apparent than with ACTH treatments, we effectively found differences in the mobility of some StAR species in the ZG between controls and rats fed a low-sodium diet for 11 days. Figure 8Go shows 2D results of a typical experiment performed four times on ZG homogenates. Compared with controls, the three 5.8/29, 5.5/29, and 5.45/29 species (arrows) were increased in the ZG under sodium restriction. Moreover, a 5.4/29 species (arrow) was found in the ZG preparations of sodium-restricted rats and not in controls.



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  		Figure 8. Effects of sodium restriction on the level of StAR in rat adrenal ZG. One group of male rats was fed a low-sodium (LOW Na+) diet for 11 days and another group was fed a normal diet and served as CTR. Analyses were performed on 50 µg of homogenate (H) proteins separated by electrofocusing and electrophoresis (2D). Western analyses were performed using a specific anti-StAR antibody. Film exposure time, 6 min. The bottom panel is a schematic that shows the mobility of the different StAR species on 2-D. Each species is expressed as pI over MW (in kDa).

 
Two-D analyses were also performed on mitochondrial preparations of control and sodium-restricted rats. Four immunoreactive bands were revealed (5.9/29, 5.7/29, 5.5/29 and 5.4/29) in the ZG mitochondrial preparations of sodium-restricted rats, and two (5.9/29 and 5.7/29) bands were revealed in controls (Fig. 9Go). Sodium restriction did not affect StAR in the ZFR (results not shown). Analyses performed on the ZG SNs show the presence of four common immunoreactive bands (5.9/29, 5.7/29, 5.4/28, and approximately 8.5/37) in both control and sodium-restricted rats. An additional band (5.5/29) was revealed only in the SN of treated animals (arrow), whereas the 5.4/28 band decreased in intensity (Fig. 10Go).



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  		Figure 9. Effects of sodium restriction on the level of StAR in rat adrenal ZG. Two groups of male rats were fed a LOW Na+ diet for 11 days, and another group was fed a normal diet and served as CTR. Analyses were performed on 50 µg of mitochondrial proteins separated by electrofocusing and electrophoresis (2D). Immunoblotting analyses were performed using a specific anti-StAR antibody. Film exposure time, 7 min. The bottom panel is a schematic that shows the mobility of the different StAR species on 2-D. Each species is is expressed as pI over MW (in kDa).

 


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  		Figure 10. Effects of sodium restriction on the level of StAR in rat adrenal ZG. Two groups of male rats were fed a LOW Na+ diet for 11 days, and another group was fed a normal diet and served as CTR. Analyses were performed on 50 µg of SN proteins separated by electrofocusing and electrophoresis (2D). Western analyses were performed using a specific anti-StAR antibody. Film exposure time, 1 h. The bottom panel is a schematic that shows the mobility of the different StAR species on 2-D. Each species is expressed as pI over MW (in kDa).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this work, we have studied the in vivo expression of StAR in adrenals of control, ACTH-treated, and sodium-restricted rats by immunofluorescence and electron microscopy, and by immunoblotting after separation of proteins by electophoresis and by isoelectric focusing/electrophoresis. The polyclonal anti-StAR antibody used was directed against the mouse mature StAR molecule (23) and was able to react in this study against rat adrenal StAR precursors and mature rat StAR.

In the present study, StAR was found predominantly in the ZG and ZF; in the ZR of human (34) and rat adrenals (32), StAR was observed in the ZR but (to varying levels) an observation also confirmed in the current study. By in situ hybridization (33), the strongest expression of StAR messenger RNA (mRNA) was located in the ZF and ZR; and 2 days after bilateral nephrectomy, all zones of the rat adrenal cortex expressed StAR mRNA to the same extent. In agreement with our results, Lo et al. (32) found StAR immunoreactive cells in the rat adrenal ZG, ZF, and ZR. The same authors (32) reported an intense immunohistochemical staining for StAR protein in the entire medulla of rat adrenal, whereas immunostaining for 3ß-hydroxysteroid dehydrogenase was observed only in the adrenal cortex. By in situ hybridization, Peters et al. (33) observed some single cells or groups of two to three cells positively stained for StAR mRNA in the medulla. By immunocytochemistry, Pollack et al. (34) observed minimal-to-moderate staining in the human adrenal cortex and no positive staining in the medulla. In our study, few cells of the medulla zone were positively stained, but we do not know whether these cells are of medullary or cortical origin. Indeed, the presence of steroidogenic cells was previously reported in the medulla, suggesting that steroids might be produced at this site (35, 36). The significance of the presence of StAR in the medulla of rat adrenal is yet to be established.

In the present study, ACTH increased the immunofluorescence signal in the ZG and the ZF, 5 h after treatment. We observed no increase in the ZR, although the fluorescent signal was still present in many dispersed cells. However, the number of positive cells seemed smaller than that of control glands. Minimal StAR staining was also reported in ZR of the human adrenal (34), indicating that their cortical zone has a weak activity in steroidogenesis.

The intermediate zone between the ZG and ZF was negative and remained as such for StAR, even after ACTH stimulation. Thus, this zone, previously described by Mitani et al. (31), would not function as a reservoir of steroidogenic cells as proposed by Peters et al. (33), at least after ACTH stimulation. The same authors suggested that the increase in ZG width, after AII stimulation, was the result of activation of previously quiescent cells and not of proliferation. Results from our laboratory (unpublished observations) confirm that the increase in ZG width, after sodium restriction, does not result mainly from proliferation of the ZG or intermediate zone cells.

At high magnification, the immunofluorescent signal appeared in the ZG adrenal cells (Fig. 1CGo) as bright dots in the cytoplasm, whereas nuclei were negative, suggesting mitochondrial localization for StAR. In agreement, the dotted appearance of the StAR fluorescent signal was also observed by Wang et al. (37) on confocal images. Ultrastructural analysis with protein A-gold clearly demonstrated that, indeed, StAR is mainly associated with MT. Moreover, in the present study, 98% of StAR antigenic sites were localized into MT. This is in agreement with results obtained in other studies where 90% of StAR antigenic sites were found in rat ovary MT (38). Over 95% of the gold particles were located inside MT in COS-1-transfected cells with a plasmid harboring StAR cDNA (37), and 96% in the rat adrenal MT (39). In the latter species, antigenic sites were localized to the inner compartments of the organelle (39) and were not confined to the mitochondrial outer membrane, where it is hypothesized to exert a biological effect related to facilitating cholesterol entry and increasing steroidogenesis. By electron microscopic studies, we also observed that the number of StAR antigenic sites increased in ZG MT, 5 h after ACTH administration, but it was not increased after 1 h. This confirms our previous results of immunoblotting analysis (16), indicating that, during this acute stimulation period and maximal steroidogenesis, which occurred within 1 h after ACTH stimulation, there was no apparent increase in the StAR mitochondrial content. This adds to the concept that the apparent entry of StAR into MT is not mandatory for an increased steroidogenesis upon ACTH stimulation. These data are in agreement with results of experiments using COS-1 cells transfected with a cDNA, producing truncated StAR proteins lacking N-terminal amino acids necessary for mitochondrial targeting. This truncated StAR was not imported into MT, as visualized by electron microscopy, but these cells were still as active as those transfected with wild-type StAR in stimulating steroidogenesis (37, 40). Furthermore, these authors (40) indicated that they saw no evidence for selective accumulation of the truncated StAR on the outer mitochondrial surface, giving no indication on the specific nature of interactions between StAR and the mitochondrial surface. In the ZF, stimulation with ACTH did not result in a significant increase in StAR accumulation in MT, neither after 1 h nor after 5 h. However, these data are misleading, because the number of gold particles was increased by 1.5-fold, when compared with controls. When looking at the high SE (Table 1Go) in the 5-h group, we reach the conclusion that the ZF of one of the animals was not responsive to the ACTH stimulation. For unknown reasons, some animals have an aberrant response to various treatments, as previously observed in other biochemical and morphological studies in our laboratory.

The effect of ACTH on StAR, observed by immunoblotting analysis, confirmed our previous report, showing (16) that in vivo ACTH administration to rats increased StAR adrenal content (Figs. 3Go and 4Go). However, in comparison with our previous report, in addition to the main 29-kDa species, the polyclonal anti-StAR antibody used in that study revealed many additional minor ACTH stimulated bands in the area comprised between 30 and 37 kDa that were not detected earlier (16). The second major immunoreactive band (30-kDa) localized exclusively into MT of the ZG and ZFR of ACTH-treated animals, as seen in Figs. 3Go and 4Go. This species was quantitatively the second in importance and appeared only 3–5 h after ACTH stimulation and, therefore, is likely to play a role in ACTH action. The same figures also show that some minor StAR species were found uniquely into MT and others uniquely into the SN of groups treated with ACTH. Results of Fig. 4Go also confirm our above cited report (16), showing that in vivo ACTH increases the StAR adrenal content after 5 h, but not after 1 h, of treatment. This observation is valid for the main and for the minor immunoreactive species. This confirms that increased steroidogenesis is not attributable to an increased quantity of StAR in the rat adrenal cortex after acute ACTH stimulation, such an increase being detected only 3–5 h after stimulation. At the present time, we have no explanation for why the 36-kDa band intensity was lowered in the ZFR but not in the ZG, 1 h after ACTH administration. This 36-kDa band is probably identical to the 37-kDa precursor reported by others (4, 37); the 1-kDa difference is within experimental error and may also be attributable to different protein standards used. The presence of many minor immunoreactive StAR bands in the area comprised between 30 and 39 kDa (Figs. 4Go and 5Go) suggests that sequential processing or posttranslational modifications may occur in vivo. It is unlikely that these protein bands are degradation products that arise from manipulations, because the intensity of these species was specifically increased upon ACTH administration. In agreement with this, COS-1 cells, transfected with a plasmid harboring the human StAR cDNA and incubated in the presence of [35S]methionine, also yielded many 35S-labeled immunoreactive bands migrating on SDS-PAGE between 29 and 37 kDa (40), suggesting sequential processing of the StAR precursor protein, as has also been proposed by Clark et al. (4).

In rat, a huge ACTH-stimulated increase in corticosterone, before any detectable increase in StAR protein or mRNA, was reported (41), indicating that ACTH had rapidly reached adrenal cell receptors after injection and had initiated a quick action on adrenal cells. We have also reported that im injection of ACTH induced a rapid increase in steroidogenesis without affecting the level of StAR (16); furthermore, the level of StAR mRNA was increased 1/2 h after ACTH administration, whereas the level of its protein was not yet increased after 1 h.

2D-PAGE and immmunoblotting analyses revealed important differences in the migration of immunoreactive StAR species in the ZG from control and ACTH-treated rats. However, these changes were much more evident 5 h, than 1 h, after ACTH treatment. As expected from 1D results, compared with control, there were not many changes 1 h after ACTH treatment, except for small changes, such as the disappearance of the 5.57/29 species. Because this difference has been observed in three separate experiments, we consider this result as real, and we thus speculate that it could contribute importantly to the mechanism of ACTH action during the first phase of acute steroidogenesis stimulation. Much more striking changes occurred in the ZG, 5 h after ACTH administration. The 5.9/29, 5.7/29, and 5.57/29 bands seemed slightly increased, whereas the 5.5/29 was greatly increased; additional acidic StAR bands were revealed at 5.8/29, 5.4/29, 5.35/29, and 5.2/29; three more basic species were more readily revealed (~6.65/31, ~8.5/34, and ~8.5/36). The latter three bands could well be the same StAR species as detected by 1D in the 31- to 39-kDa area (Figs. 4Go and 5Go) and could correspond to StAR precursors. Although less important in quantity, we have considered bands 5.8/29 and 5.57/29 as distinct StAR species, based on the fact of their absence in control preparations. The calculated pIs of the rat StAR precursor protein and of the mature proteins are 8.80 (284 AA), 7.32 (246 AA), 7.32 (244 AA), and 7.80 (241 AA), respectively (calculated according to the rat StAR sequence (42) and putative cleavage sites as reported). This suggests that StAR species with basic pI in the area of 8.5 are not phosphorylated StAR precursors. This is in agreement with the results of King et al. (39), indicating that the mouse 37-kDa precursor was not phosphorylated, because its pI was not changed after alkaline phosphatase digestion.

In the experiment using the whole adrenal, the separation of MT from SN revealed that some StAR species were distributed exclusively into MT and others into the SN. Compared with controls, the mitochondrial preparation of ACTH-stimulated adrenals possessed more 29-kDa species and more acidic bands than controls. Furthermore, a 30-kDa species with a pI of 5.78 was found exclusively in MT of experimental animals; this major band probably corresponds to the induced 30-kDa band revealed by 1D as seen in MT, 5 h after ACTH treatment (Figs. 3Go and 4Go). The SN content also revealed differences between ACTH-treated and control groups. ACTH induced two (5.5/29 and 5.4/29), whereas a decrease in the intensity of a 5.4/28 was observed. Moreover, a species of approximately 8.5/36 kDa was more readily seen in SNs than in MT, indicating that this StAR species, probably a StAR precursor, is located outside MT. In agreement with our results, King et al. (39) reported that there was no StAR precursor in the mitochondrial fraction, whereas the level of four 30-kDa mature forms was enriched. We therefore speculate that changes occurring at the SN level are important for the action of ACTH in maintaining an increased steroidogenesis in rat adrenal.

We found differences between mitochondrial preparations originating from the ZG and those from the ZFR, especially in the area of pI 5.5 and 5.3. Some acidic forms revealed in the ZFR MT were absent in the ZG (Fig. 7Go), indicating that StAR may be processed differently in the ZG than in the ZFR.

The pI values of the different StAR species reported in this study are slightly different from those recently reported by Ariyoshi et al. (41), which found two 30-kDa StAR proteins in adrenals of rats with pI of 6.5 and 6.6, and an additional pair of low pI StAR form (pI 6.2–6.3) upon ACTH stimulation; pI of 6.5 and 6.3 were reported by Pon and Orme-Johnson (43) for [35S]methionine-labeled proteins now identified as StAR. These entities most probably correspond to the four main StAR species (pI 5.9, 5.7, 5.5, and 5.4) that we are reporting in this study. Differences in pI values are most probably caused by the different standards or ampholytes used.

Surprisingly, sodium restriction for 11 days, which increases circulating renin activity (17), and consequently AII availability, to stimulate adrenocortical cells, provoked increased circulating aldosterone (results not shown) but had much less effect on the adrenal StAR content than ACTH treatments. As previously reported (17), when rats were fed a low-sodium diet for 2 days, plasma aldosterone levels were increased, but the quantity of adrenal StAR, as measured by 1D-PAGE, was comparable with that of control (data not shown). Even at shorter term (after 1 day of sodium restriction), though PRA was not statistically increased [although plasma aldosterone levels were increased (17)], the quantity of StAR in the ZG and the ZFR was not different from controls. This indicates that, even during its acute stimulatory phase, sodium restriction seems to have quantitatively less effect on StAR than ACTH. Changes occurred, however, in the ZG of sodium-restricted rats but not in the ZFR. It is not surprising that sodium restriction had no effect on the ZFR because these zones of the adrenal cortex are not responsive to AII (1). When analyzing results obtained for the ZG homogenates (Fig. 8Go), we observed at least four differences induced by the sodium restriction, compared with controls. The 5.8/29, 5.5/29, and 5.45/29 bands increased in intensity; a new band appeared at 5.4/29, whereas the intensity of the 5.4/28 band decreased. When MT and SN were separately analyzed, the 5.5/29 and 5.4/29 bands were found in MT (Fig. 9Go), whereas only the 5.5/29 was found in the SN (Fig. 10Go); the 5.4/28 band was also found in SNs and was decreased in preparations of sodium-restricted animals. Other basic bands (~8.5/36), seen mainly in SNs, correspond mostly to the precursor protein. Differences observed in the ZG between groups of sodium-restricted and control rats are small but real, because they were found in three different experiments. Similar results were obtained after 2 days of sodium restriction (results not shown). These results indicate that sodium restriction produced small, but physiologically important, changes on StAR of the ZG to sustain elevated aldosterone synthesis. Sodium restriction activates the renin angiotensin system (1), and the effect of AII on StAR in rat adrenal glomerulosa cells has been reported by Elliot et al. (21); these authors reported that AII treatment to rat adrenal glomerulosa cells increased the incorporation of [35S]methionine into two 28.5-kDa proteins. Hartigan et al. (20) also reported the enhancing effect of AII on [35S]methionine incorporation into a protein pp30 in rat adrenal ZG cells. AII was also shown to increase StAR when added to NCI H295 cell cultures (19).

Taken all together, these results indicate that StAR does not quantitatively change during acute stimulation by ACTH, at least during the first hour after treatment; and consequently, to explain the action of StAR during this acute stimulation period, one should look at changes of other StAR characteristics. Indeed, in two situations where adrenal steroidogenesis is enhanced, 1 h after ACTH administration to rats and in sodium-restricted animals, only small changes in StAR characteristics were observed; and these changes were qualitative rather than quantitative. In the ZG homogenate, changes on StAR that occurred in those two situations were on species migrating in the area comprised between species 5.7/29 and 5.4/29. These changes, however, were not identical for ACTH and sodium-restriction groups, indicating different mechanisms of action between these two stimulating factors. Changes also occurred in the 5.7/29 and 5.4/29 area in the SN of treated rats 5 h after ACTH stimulation.

Thus, under physiological conditions, such as when rats are conditioned to sodium restriction for many days, or at the beginning of ACTH stimulation (up to at least 1 h after treatment but not 3 h after, in experiments described in this article), we speculate that qualitative (instead of quantitative) changes in StAR are necessary for its steroidogenic action. In other words, under physiological conditions, only small changes in StAR might be necessary to stimulate steroidogenesis. In fact, as reported by Arakane et al. (40), StAR exerts its action at nanomolar, and maybe even at lower, molar concentrations. These results are also arguing in favor of looking for small changes instead of large changes occurring during StAR processing, to comprehend the mode of action of this molecule in acutely stimulated adrenal steroidogenesis.

The accumulation of StAR into MT, occurring 3–5 h after ACTH administration, might not be required to increase steroidogenesis but should rather be considered as a way for adrenocortical cells to terminate or dispose of this molecule once its action is completed. Others (37, 40) who showed that truncated StARs, which do not enter into MT, still have high steroidogenic activity also suggested this hypothesis. Although we have not provided data showing that the protein species identified in the present study are phosphorylated forms of StAR, the fact that many 29-kDa StAR species found into mitochondrial preparations are acidic, and to varying degrees, suggests that this molecule might be phosphorylated at many different positions. Hence, it is speculated that phosphorylation can either increase, as previously shown (22), or perhaps decrease, StAR activity (depending on the site of phosphorylation and on the number of phosphorylated sites on the protein). Further studies will be necessary to comprehend the role of StAR accumulation inside mitochodria occurring 3–5 h after ACTH stimulation in vivo.


   Footnotes
 
1 This work was supported by a grant from the Medical Research Council of Canada (MT-10983) and by the Heart and Stroke Foundation of Canada (to J.-G.L.) and by HD-27571 and HD-35544 (to D.B.H.). Back

2 Preliminary results were communicated at the 8th Symposium of the Adrenal Cortex (24 ). Back

Received February 19, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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