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Endocrinology Vol. 138, No. 12 5415-5426
Copyright © 1997 by The Endocrine Society

ARTICLES

Ex Vivo Regulation of Adrenal Cortical Cell Steroid and Protein Synthesis, in Response to Adrenocorticotropic Hormone Stimulation, by the Ginkgo biloba Extract EGb 761 and Isolated Ginkgolide B1

Hakima Amri, Katy Drieu and Vassilios Papadopoulos2

Departments of Cell Biology (H.A., V.P.) and Pharmacology (V.P.), Georgetown University Medical Center, Washington, D.C. 20007; and Institut Henri Beaufour-IPSEN (K.D.), 75116 Paris, France

Address all correspondence and requests for reprints to: Dr. V. Papadopoulos, Department of Cell Biology, Georgetown University Medical Center, 3900 Reservoir Road, Washington, D.C. 20007. E-mail: papadopv{at}gunet.georgetown.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We previously demonstrated that repeated treatment of rats with the standardized extract of Ginkgo biloba leaves, EGb 761, and its bioactive component ginkgolide B (GKB), specifically reduces the ligand binding, and protein and messenger RNA expression of the adrenal mitochondrial peripheral benzodiazepine receptor (PBR), a key element in the regulation of cholesterol transport, resulting in decreased circulating corticosterone levels. Adrenocortical cells were isolated from rats treated with EGb 761 or GKB and cultured for 2 and 12 days. The effect of ACTH on normal and metabolically labeled cells was examined. Corticosterone levels were measured by RIA, and protein synthesis was analyzed by two-dimensional gel electrophoresis. Ex vivo treatment with EGb 761 and GKB resulted, respectively, in 50% and 80% reductions of ACTH-stimulated corticosterone production by adrenocortical cells cultured for 2 days compared with that by cells isolated from saline-treated rats. Two-dimensional gel electrophoresis analysis revealed that in cells from both control and drug-treated animals, ACTH induced the synthesis, at the same level, of a 29-kDa and pI 6.4–6.7 protein identified as the adrenal steroidogenic acute regulatory protein (StAR). In addition, treatment with EGb 761 and GKB specifically altered the synthesis of seven proteins, including inhibition of synthesis of a 17-kDa, identified as PBR. After 12 days in culture, ACTH-stimulated adrenocortical cell steroid synthesis was maintained, and it was identical among the cells isolated from animals treated with GKB or saline. Under the same conditions, the expression of PBR was recovered, whereas no effect of ACTH on the 29-kDa and 6.4–6.7 pI protein (StAR) or other protein synthesis could be seen. A comparative analysis of the effects of GKB and EGb 761 on adrenocortical steroidogenesis and protein synthesis identified, in addition to the 17-kDa PBR, target proteins of 32 kDa (pI 6.7) and 40 kDa (pI 5.7–6.0) as potential mediators of the effect of EGb 761 and GKB on ACTH-stimulated glucocorticoid synthesis. In conclusion, these results 1) validate and extend our previous in vivo findings on the effect of EGb 761 and GKB on ACTH-stimulated adrenocortical steroidogenesis, 2) demonstrate the specificity and reversibility of EGb 761 and GKB treatment, 3) question the role of the 29-kDa, 6.4–6.7 pI protein (mature StAR) as the sole mediator of ACTH-stimulated steroid production, and 4) demonstrate the obligatory role of PBR in hormone-regulated steroidogenesis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
TROPHIC hormone regulation of steroid synthesis can be either acute, occurring within minutes and resulting in rapid steroid synthesis, or chronic, occurring within hours and resulting in the continued synthesis of steroids reflecting increased synthesis of the components of the steroidogenic machinery. The primary point of control in the acute stimulation of steroidogenesis by hormones involves the first step in this biosynthetic pathway, where cholesterol is converted to pregnenolone by the cholesterol side-chain cleavage cytochrome P-450 (P-450scc) and auxiliary electron-transferring proteins, localized on inner mitochondrial membranes (1, 2). More detailed studies have shown that the reaction catalyzed by P-450scc is not rate determining in the synthesis of steroid hormones, but, rather, it is the transport of the precursor, cholesterol, from intracellular stores to the inner mitochondrial membrane where steroid production begins (1, 2). Although this hormone-dependent transport mechanism was localized in the mitochondrion (1, 2), identification of the components responsible for this transport mechanism remains a challenge.

In search of this mechanism, we identified the peripheral-type benzodiazepine receptor (PBR) as a key regulatory element in this process (3, 4). PBR was originally discovered because it binds the benzodiazepine diazepam with relatively high affinity (3). However, PBR is a class of binding sites for benzodiazepines pharmacologically and biochemically distinct from the well characterized {gamma}-aminobutyric acid receptors found in the central nervous system. PBR was found to be particularly high in steroid-producing tissues (3), where it was primarily localized in outer mitochondrial membrane (5). An 18-kDa isoquinoline-binding protein was identified and characterized as PBR (6, 7, 8). It was then demonstrated that PBR is a functional component of the steroidogenic machinery (9, 10, 11, 12, 13) mediating cholesterol delivery from outer to inner mitochondrial membrane (14). Targeted disruption of the PBR gene in steroidogenic cells resulted in the inhibition of cholesterol transport to the inner mitochondrial membrane and arrest of steroid biosynthesis, further supporting the critical role of PBR in the regulation of steroidogenesis (4). Moreover, molecular modeling studies suggested that PBR may be an outer mitochondrial membrane channel/transporter for cholesterol (4).

In addition to PBR, other proteins were shown to be involved in the hormonal regulation of steroidogenesis (2), including a 28- to 30-kDa and 6.4–6.7 pI protein named steroidogenic acute regulatory protein (StAR) (15, 16, 17). StAR has been shown to be expressed in response to trophic hormones, and it has been suggested to be the major mediator of hormone-stimulated steroidogenesis (15, 16, 17).

First, the published reports on the pathogenic potential of glucocorticoid excess on neural development, aging, and neurological diseases related to hippocampal damage (18, 19, 20, 21, 22, 23), and second, the numerous studies describing the beneficial effects of Ginkgo biloba extract on cognitive functions and adaptation (24, 25, 26, 27, 28, 29), two processes negatively affected by glucocorticoid excess (19, 20), brought us on the serendipitous finding that G. biloba extract treatment of rats brought circulating glucocorticoids close to basal levels (30). In these studies, we used a standardized extract of G. biloba leaves, termed EGb 761, and isolated ginkgolides (30). In search of the mechanism of action of these substances, we identified PBR as their molecular target in the adrenal tissue. Repeated treatment of rats with EGb 761 and ginkgolide B (GKB) reduced adrenal PBR messenger RNA, protein, and ligand binding expression and, thus, cholesterol substrate availability to P450scc without affecting P450scc activity (30). In these studies, the GKB-induced decrease in PBR expression and corticosterone synthesis resulted in increased circulating ACTH levels and the expected induction of the endogenous PBR ligand, the polypeptide diazepam binding inhibitor (31), and StAR protein (16). Although this finding demonstrated that StAR is regulated by ACTH in vivo, it questioned the role of this protein in the induction of the steroidogenic process in the absence of PBR. A summary of these data are presented in Fig. 1Go.



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  		Figure 1. Summary of the endocrine and molecular responses to EGb 761 and GKB in vivo treatment. Male 80-day-old Sprague-Dawley rats were treated either orally (by gavage) with saline solution (0.9% NaCl) or with EGb 761 extract (100 mg/kg) for 8 days or were injected (ip route) with GKB (2 mg/kg). Animals were killed on day 9, and adrenal glands, serum, and plasma were collected. Corticosterone and ACTH were assessed by RIA. P450 activity was determined in the mitochondrial subcellular fraction in the presence or absence of 20{alpha}-hydroxycholesterol. PBR binding was performed using [3H]PK 11195 as a ligand. The amounts of 18-kDa PBR protein were quantified by immunoblot analysis after SDS-PAGE fractionation of adrenal mitochondrial proteins. The levels of the 18-kDa PBR messenger RNA were determined by RNA (Northern) blot analysis. Diazepam binding inhibitor and StAR protein levels were assessed by immunocytochemistry followed by image analysis. The data are presented as a percentage of the response/changes observed. For more details, see Ref. 30.

 
In the present report we investigated the specificity of the in vivo effect of EGb 761 and GKB on circulating glucocorticoid levels on the in vitro adrenocortical cell protein and steroid synthesis in response to ACTH. Our goal was to determine whether, in addition to PBR, there is another protein target(s) of these substances and, thus, to define their role in the ACTH-dependent process responsible for the induction of steroidogenesis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
The extract of G. biloba, EGb 761 (lot K923), and GKB (BN 52021) isolated from EGb 761 (24) were provided by the Institute Henri Beaufour-IPSEN (Paris, France). [1,2,6,7-N-3H]Corticosterone (SA, 80.00 Ci/mmol) and [7-N-3H]pregnenolone (SA, 21.1 Ci/mmol) were obtained from DuPont-New England Nuclear (Wilmington, DE). Translabeled L-[35S]methionine, L-[35S]cysteine ([35S]met/cys; SA, >1000 Ci/mmol), DMEM deficient in methionine and cysteine, as well as anticorticosterone and antipregnenolone antisera were purchased from ICN (Costa Mesa, CA). N6,2'-O-Dibutyryl cAMP [(Bu)2cAMP] (dbc AMP), collagenase type IA, deoxyribonuclease (DNase) type III, {alpha}-tocopherol ascorbic acid, selenium, and MEM amino acids and vitamins were obtained from Sigma Chemical Co. (St. Louis, MO). ACTH-(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24) was obtained from Peninsula Laboratories (Belmont, CA). PBS (pH 7.2) and cell culture supplies were purchased from Life Technologies (Grand Island, NY). Horse serum was obtained from BioWhittaker (Walkersville, MD). FBS and penicillin/streptomycin were purchased from Biofluids (Rockville, MD). DMEM-Ham’s F-12 medium was supplied by Irvine Scientific (Santa Ana, CA). Cell culture plasticware was obtained from Corning (Corning, NY). Electrophoresis reagents and materials were supplied by Bio-Rad (Richmond, CA). Nitrocellulose (0.45 µm) and polyvinylidene difluoride (PVDF) were obtained from Hoefer Scientific (San Francisco, CA). Glass fiber GF/C filters were purchased from Whatman (Clifton, NJ). All other chemicals of analytical quality were obtained from various commercial sources.

Animals and experimental design
Male 80-day-old Sprague-Dawley rats were obtained from Charles River Breeding Laboratories (Wilmington, MA). Rats were housed at the Georgetown University Research Resources Facility under controlled light and temperature, with free access to rat chow and water. They were housed in groups of three and acclimated to their new conditions for 4 days before treatment. All experimental protocols were reviewed and approved by the Georgetown University animal care and use committee. Saline (0.9% NaCl in double-distilled water) and EGb 761 (100 mg/kg) were administered orally (gavage) every day for a total of 8 days, and rats were killed 24 h after the last treatment. GKB (2 mg/kg) was administered by ip injection every day for a total of 8 days, and the animals were also killed 24 h later. All compounds were dissolved in double distilled water, and treatments were performed at 1000 h. The length of treatment was based on our previous observations (30). The rational for the concentrations used and the chosen routes of administration have been presented previously (30).

Steroid measurements
Serum corticosterone was measured by RIA as previously described (30). The levels of corticosterone and pregnenolone were also measured in the culture medium by RIA. Analysis of the RIA data were performed using the IBM-PC RIA Data Reduction program (version 4.1) obtained from Jaffe and Associates (Silver Spring, MD).

Preparation of adrenal cell and culture
Adrenal glands from control and treated rats were collected after laparotomy. Rat adrenal cells were prepared according to the methods described by Dibartolomeis and Jefcoate (32) and Arai and Widmaier (33) with modifications. In brief, adrenals were washed with PBS buffer, septically removed from surrounding fat, and decapsulated. The tissue was minced in a small volume of PBS buffer, washed several times with the same buffer, and then transferred into a 50-ml sterile capped tube. The tissue fragments were allowed to settle, and the PBS buffer was removed and replaced with 20 ml fresh DMEM-Ham’s F-12 medium supplemented with HEPES (20 mM). Medium containing collagenase type 1A (2.5 mg/ml) and DNase 1 type III (0.1 mg/ml) was filter sterilized (Millipore Corp., Bedford, MA; 0.45 µm) and adjusted to pH 7.2. The minced tissue was then incubated in this medium for 30 min at 37 C in a shaking water bath. The supernatant was carefully removed without disturbing the settled tissue. The remaining tissue fragments were resuspended in 10 ml medium and pipetted up and down several times. The supernatant was collected, and the remaining tissue fragments were resuspended in 20 ml medium containing collagenase and DNase, as described above, and submitted to an additional 30-min incubation. All supernatants were then pooled and centrifuged at 2000 rpm for 5 min. The cells collected were resuspended and washed with culture medium supplemented with 10% horse serum, 2.5% FBS, 100 IU/ml penicillin, 100 mg/ml streptomycin, 1 mM {alpha}-tocopherol, 100 mM ascorbic acid, 50 nM selenium, and MEM amino acids and vitamins. Cells were plated either in 35-mm multiwell plates or in the 100-mm plates at a cellular density ranging between 0.5–1.4 x 106 cells/well and maintained at 37 C in a humidified atmosphere of 95% air-5% CO2. The medium was removed and replaced with fresh medium 24 h after incubation.

Incubation of cells with [35S]met/cys and sample solubilization
Rat adrenal cortical cells (1.5 x 106/well) were translabeled with [35S]met/cys (0.6 mCi/well) in met/cys-free medium. After 2-h incubation, ACTH (10 ng/ml) was added, or not, to the cells, and the incubation was carried out for an additional 3 h. In some experiments, the cAMP analog, (Bu)2cAMP (1 mM), was used to stimulate the cells. The sample preparation for two-dimensional electrophoresis (2D-PAGE) was performed according to the procedure described below. The efficiency of the radiolabeled incorporation was determined as described by Pon and Orme-Johnson (34). In brief, aliquots of 3 µl were precipitated by 100 times the volume of 10% trichloroacetic acid. The precipitate was heated at 90 C for 20 min in a water bath and allowed to stand at 4 C overnight. The samples were filtered on glass fiber filters, rinsed with 5% trichloroacetic acid, and washed with ethanol. The filters were air-dried, and radioactivity was determined by liquid scintillation spectrometry.

2D-PAGE
2D-PAGE was performed according to the method of O’Farrell (35) by us and Kendrick Laboratories (Madison, WI) as follows: Isoelectric focusing was carried out in glass tubes of 2 mm inner diameter using 2% Resolytes pH 4–8 ampholines (BDH, Hoefer Scientific Instruments, San Francisco, CA) for 9600 V-h (volt-hours). After equilibration for 10 min in SDS sample buffer (10% glycerol, 50 mM dithiothreitol, 2.3% SDS, and 0.0625 M Tris, pH 6.8), the tube gels were sealed to the top of 12.5% acrylamide slab gels (0.75 mm thick), and SDS slab gel electrophoresis was carried out for about 4 h at 12.5 mA/gel. The following 14C molecular mass markers were added to a well in the agarose that seals the tube gel to the slab gel: myosin, 200 kDa; phosphorylase b, 97.4 kDa; BSA, 69 kDa; ovalbumin, 46 kDa; carbonic anhydrase, 30 kDa; and lysozyme, 14.3 kDa. These markers appear as bands at the basic edge of the x-ray films. The slab gels were fixed in a solution of 10% acetic acid and 50% methanol overnight. The next day the gels were rehydrated in 10% acetic acid for 1 h and then dried onto filter paper with the acid end to the left. Autoradiography was carried out using Kodak X-Omat AR film (Eastman Kodak, Rochester, NY) with an exposure of 1 day. The x-ray films were scanned at 200-µm resolution using a LGS-50 Laser Scanning Densitometer (Digital Instruments Corp., Newark, DE) calibrated for linearity with a Mells Griot Neutral Density Filter Set (Mells Griot, Irvine, CA). The digitized images were formatted so that 0–3.0 OD units were expanded to full scale. The major polypeptide spots were automatically detected and quantified using Phoretix 2D Full software with Spot Detection Parameters as follows: sensitivity, 250; operator size, 31; number of edge pixels, 5; center box size, 5; and background factor, 50.

Immunoblot procedure
Proteins were separated by 2D-PAGE as described above. The slab gel was transferred to transfer buffer (12.5 mM Tris, pH 8.8; 86 mM glycine; and 10% methanol) and transblotted onto a PVDF membrane overnight at 200 mA. The proteins used as molecular mass standards are similar to those described above. The PVDF membrane was stained with Coomassie brilliant blue and then rinsed extensively in ultrapure water. Before the immunoblotting, the membrane was washed briefly in methanol and incubated with anti-PBR antiserum (30) (1:2000) or anti-StAR antiserum (30) (1:1000) followed by the horseradish peroxidase-conjugated goat antirabbit second antibody. The immunoreactive proteins were revealed by chemiluminescence using the ECL detection reagent (DuPont-New England Nuclear). Proteins were also fractionated by one-dimensional SDS-PAGE, transferred onto nitrocellulose, and submitted to immunoblot analysis using the anti-PBR antibody as described above. Densitometric analysis of the immunoreactive protein bands was performed using the Sigmagel software (Jandel Scientific, San Rafael, CA).

Protein measurement
Microgram amounts of protein were quantified by the dye binding assay of Bradford (36) with {gamma}-globulin as standard.

Statistics
The results shown represent the mean ± SD or SEM from 2–6 independent experiments. The number of animals used varied from 5–10 rats/treatment·experiment. For the in vitro studies and the assays within each experiment, each point is the mean of triplicate determinations. Statistical analysis was performed by ANOVA followed by the Student-Newman-Keuls test or Dunnett’s multiple comparisons test using the Instat (v.2.04) package from GraphPad (San Diego, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The in vivo effects of G. biloba extract EGb 761 and its purified component GKB on the endocrine system of the rat were previously examined (30). These studies revealed an adrenal tissue-specific effect of these substances, resulting in a reduction of the peripheral glucocorticoid levels (30). The molecular target of EGb 761 and GKB was identified as the mitochondrial PBR. The results from these studies, represented as a percentage of the response seen, are summarized in Fig. 1Go. In the present study, we examined the effect of the in vivo treatment of rats with EGb 761 and GKB on the in vitro adrenal cortical cell steroid and protein synthesis (ex vivo studies). In our previous work we used both the isolated ginkgolide A (GKA) and GKB (30). Because the effects of these two compounds on PBR and corticosterone production were similar, we used only GKB in the present studies.

Ex vivo effect of EGb 761 and GKB on steroid production by 2-day-old rat adrenocortical cell cultures
Isolated adrenocortical cells (1.5 x 106 cells/well) were cultured for 2 days and then stimulated, or not, with ACTH for 3 h. Figure 2Go shows that cells prepared from animals treated with saline are highly responsive to increasing concentrations of ACTH. Considering that the basal value for corticosterone (100%) was about 4.8 ng/mg protein, a 40-fold stimulation was obtained with 10 ng/ml ACTH. In vivo treatment of the rats with EGb 761 resulted in a 50% inhibition of the responsiveness of the cells to high concentrations of ACTH by 50%. A more robust inhibition (80%) of the ACTH-stimulated steroidogenesis was observed using adrenocortical cells prepared from GKB-treated animals. Thus, these data further validate our previous findings on the effects of EGb 761 and GKB on circulating glucocorticoid levels.



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  		Figure 2. Effects of increasing concentrations of ACTH on corticosterone synthesis by adrenocortical primary cells obtained from animals treated with EGb 761, GKB, or saline. Adrenals were collected from rats treated as described in Fig. 1Go. Adrenocortical cells were prepared as described in Materials and Methods. Cells (0.5 x 106 cells/well) were incubated for 3 h with ACTH, and the medium was collected to measure corticosterone production by RIA. The values represent the mean ± SEM from three independent experiments (n = 8).

 
Ex vivo effect of EGb 761 and GKB on protein synthesis in 2-day-old rat adrenocortical cell cultures
Because of the close correlation between the ex vivo and in vivo effects of EGb 761 and GKB on adrenal steroidogenesis, we decided to perform a detailed study of the effects of these treatments on adrenocortical protein synthesis to identify the potential protein candidate(s) mediating this effect. Two days after isolation, adrenocortical cells were incubated with [35S]met/cys for 2 h and then stimulated, or not, with ACTH or (Bu)2cAMP for 3 h. Because similar results were obtained using ACTH or (Bu)2cAMP, only the data generated using ACTH are shown. Figure 3Go shows the autoradiograms obtained after 2D-PAGE analysis followed by autoradiography. Over 100 proteins were translabeled with [35S]met/cys. After careful examination, we identified seven proteins that were affected by treatment either with EGb 761 and GKB or with ACTH. The changes in the synthesis of these proteins are shown in Fig. 4Go and summarized in Table 1Go, following a left to right numbering according to the increasing pI from acidic to basic. Protein 1 (pI 5.7–6; 40 kDa), which appears as two distinct spots, was induced by ACTH in cells obtained from saline-treated rats. However, this stimulatory effect of ACTH could not be seen in cells obtained from rats treated with EGb 761 or GKB. These former compounds, however, induced the expression of protein 2 (pI 6.2; 17 kDa). Protein 2 can be seen in the presence and absence of ACTH. In the range of 29 kDa and at a pI ranging from 6.4–6.7, the induction by ACTH of two spots was observed (protein 3). Considering the molecular size and pI of this protein and previous findings concerning the induction of this protein by ACTH in adrenal cells (15, 34), this protein has characteristics identical to those of the adrenal StAR protein. Further immunoblot analysis (Fig. 5Go) after 2D-PAGE of radiolabeled samples demonstrated that one of the two spots of protein 3 was recognized by the anti-StAR antiserum that we developed (30). Protein 3 (StAR) was not affected by treatment with either EGb 761 or GKB. The expression of proteins 4 (pI 6.7; 32 kDa) and 5 (pI 6.8; 85 kDa) was induced by ACTH. However, treatment of the animals with EGb 761 or GKB greatly reduced or inhibited the effect of ACTH on these proteins. A series of four spots at a molecular size of 90 kDa (protein 6; pI 6.8–7.1) was induced by ACTH. A dramatic reduction, however, of the spot intensity was noticed in cells obtained from EGb 761- and GKB-treated animals. Treatment with both EGb 761 and GKB inhibited expression of a 17-kDa protein (protein 7; pI 8). The expression of this protein was not affected by treatment with ACTH. Protein 7 was identified as the PBR protein using immunoblot analysis after 2D-PAGE of the same samples used for the metabolic labeling studies, as described in Materials and Methods (Fig. 4Go, p7). The areas containing the seven spots described in this paragraph were enlarged and focused, as shown in Fig. 4Go.



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  		Figure 3. Effect of EGb 761 and GKB treatment on isolated adrenocortical cells translabeled with [35S]met/cys in the presence or absence of ACTH. Isolated adrenocortical cells were cultured for 2 days as described in Materials and Methods. Cells (1.5 x 106/well) were then incubated with [35S]met/cys (0.6 mCi/well) for 2 h and incubated with or without a saturating concentration of ACTH (10 ng/ml) for 3 h. 2D-PAGE analysis of the radiolabeled rat adrenal cells (the loads were normalized to 3 x 106 dpm) was carried out as described in Materials and Methods. Arrows denote the positions corresponding to proteins of interest. Increasing pI values from acidic to basic are shown from the left to the right edge of the gel.

 



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  		Figure 4. Enlarged autoradiograms of the seven most affected proteins by EGb 761 and GKB treatment and/or by ACTH. The arrows point to the different affected proteins 1–7. The PBR immunoblot carried out after separation of proteins (loads normalized to 30 µg) by 2D-PAGE as described in Materials and Methods. The immunoreactive 17-kDa PBR protein is denoted by the arrowhead.

 

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  		Table 1. Comparison of 35S-Met/Cys-labeled proteins in control and ACTH-treated cells isolated from saline-, EGb 761-, and GKB-treated rats

 


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  		Figure 5. Identification of the pI 6.7, 29-kDa protein as the mature form of StAR. Adrenocortical cells were labeled with [35S]met/cys (0.6 mCi/well) for 2 h and incubated with (+dbcAMP) or without (-dbcAMP) 1 mM of the cAMP analog. Enlarged autoradiograms of the cAMP-induced pI 6.4–6.7, 29-kDa protein are shown in the top and middle panels. In the lower panel, the immunoblot analysis of a (Bu)2cAMP-treated sample after separation of the proteins (150 µg) by 2D-PAGE is shown. The arrowhead points to the pI 6.7, 29-kDa immunoreactive StAR.

 
Ex vivo effect of EGb 761 and GKB on PBR protein expression in 2-day-old rat adrenocortical cell cultures
The effect of EGb 761 and GKB treatment on 17-kDa PBR protein expression was further confirmed by immunoblot analysis of cell extracts using the anti-PBR antiserum (14) (Fig. 6Go). Image analysis of the 17-kDa immunoreactive protein indicated 70% and 82% decreases in protein expression by EGb 761 and GKB, respectively.



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  		Figure 6. PBR protein expression. Solubilized rat adrenal cell extracts (40 µg/well) were fractionated by SDS-PAGE, and an immunoblot analysis was performed as described in Materials and Methods.

 
Ex vivo effect of GKB on steroid production by 12-day-old rat adrenocortical cell cultures
In our previous studies we demonstrated that the inhibitory effect of in vivo treatment with EGb 761 and GKB on adrenal PBR expression and steroidogenesis was completely reversed 11 days after the end of the treatment (30). Thus, we examined whether the ex vivo effect of these compounds on adrenocortical steroidogenesis and protein synthesis was still present in 12-day-old adrenal cortical cell cultures. Because the effect of GKB on ACTH-stimulated steroidogenesis was more pronounced than that of EGb 761, and as both substances had similar effects on adrenocortical protein synthesis, we used cells from saline- and GKB-treated animals only for these experiments. Cells were prepared as described in Materials and Methods, maintained in culture for 12 days, and then stimulated for 3 h with or without 10 ng/ml ACTH. Figure 7Go shows that 12-day-old adrenal cortical cell cultures maintained their responsiveness to ACTH; a 10-fold stimulation of corticosterone production was seen. Moreover, Fig. 7Go clearly shows that there was no difference in the ACTH-stimulated steroidogenesis between cells obtained from saline- and GKB-treated animals. In addition to corticosterone, pregnenolone production by these cells was identical among those from saline- and GKB-treated rats (Fig. 7Go). Thus, the ex vivo effect of GKB on ACTH-stimulated steroidogenesis was reversed.



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  		Figure 7. Recovery of steroid synthesis in primary adrenocortical cells after 12 days of culture. Cells were prepared from saline- and GKB-treated rat adrenals and maintained in culture for 12 days. Cells were washed and incubated with or without 10 ng/ml ACTH for 3 h. At the end of the incubation, media were collected for steroid measurements. Results shown represent the mean ± SD from a representative experiment (n = 3).

 
Ex vivo effect of GKB on protein synthesis in 12-day-old rat adrenocortical cell cultures
We then examined the profile of 12-day-old adrenocortical cell proteins by [35S]met/cys incorporation and 2D-PAGE in saline- and GKB-treated rats. Figure 8Go shows the autoradiograms from these experiments. The profiles are very similar. We further examined the fate of each of the seven previously identified proteins (Figs. 8Go and 9Go and Table 1Go). The expression of proteins 1 (pI 5.7; 40 kDa), 4 (pI 6.7; 32 kDa), and 7 (pI 8; 17 kDa; PBR) was completely recovered. Protein 2 (pI 6.2; 19 kDa) was induced by GKB, but when stimulated, it disappeared with ACTH. Protein 3 (pI 6.4–6.7; 29 kDa; StAR) was absent from all cells, and its expression was not induced by ACTH. Proteins 5 (pI 6.8; 85 kDa) and 6 (pI 6.8–7.1; 90 kDa) were not altered by the treatment with GKB or ACTH. An additional protein, protein 8 (pI 6.6; 15 kDa) was regulated by GKB and ACTH, but not in a coordinated fashion. These data demonstrate that in addition to PBR (protein 7), the expression of proteins 1 (pI 5.7–6.0, 40 kDa) and 4 (pI 6.7, 32 kDa) is modulated by treatment with EGb 761 and GKB, and suggest and these proteins may mediate the effects of these substances on ACTH-stimulated steroidogenesis.



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  		Figure 8. Effect of GKB treatment on protein synthesis, examined by [35S]met/cys incorporation, by 12-day-old cultured adrenocortical cells in the presence or absence of ACTH. Two-dimensional gels (loads were equalized to 780,000 dpm) were run as described in Fig. 3Go. The arrows point to the proteins affected by saline or GKB treatment. The arrowheads indicate the PBR protein location and the expected location of StAR.

 


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  		Figure 9. Enlarged autoradiograms of the specific proteins described in Fig. 7Go. The arrows point to the various affected proteins 1–8.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The G. biloba extract EGb 761 is widely used for its medicinal effects on vascular insufficiency and neurosensory problems and its beneficial effects on disturbances in vigilance, memory, mood changes, adaptation to daily stressors, and loss of cognitive functions associated with aging (24, 25, 26, 27, 28, 29). In addition to these actions, recent studies demonstrated that repeated oral administration of EGb 761 exerted antistress effects in rodent animal models (24, 25, 27, 28, 29). This antistress activity of EGb 761 was then shown to be distinct from the classical antidepressant and anxiolytic action (28, 29). Considering that a major consequence of stress is the dramatic rise in serum glucocorticoid levels (37), and the findings that chronic stress or exposure to high concentrations of glucocorticoids induced hippocampal damage which could be reduced by adrenalectomy, we advanced the hypothesis that a potential mechanism of action of EGb 761 is the regulation of glucocorticoid synthesis. This hypothesis was tested, and the results obtained showed that indeed an 8 day treatment of adult rats with the extract EGb 761 (24) resulted in a decrease in serum corticosterone levels (30). The terpenoid GKB, one of the bioactive components of EGb 761, was shown to exert a similar inhibitory effect on corticosterone biosynthesis, although more pronounced because of its better absorption when administered iv (30).

During these studies we noted that treatment with EGb 761 decreased serum corticosteroid levels by 50%. Thus, there is enough corticosterone remaining to support the glucocorticoid-dependent functions. This is also supported by the observations that treatment with EGb 761 does not exert any detrimental effect on animal and human health (24, 25, 26, 27, 38). Moreover, we should also consider the possibility that 50% of the measured corticosteroid levels may reflect the normal "unstressed" condition. Surveying the literature for circulating corticosterone levels in rats, we found that there is a large variation in the reported values ranging from 4 to 40 ng/ml. In addition to the well known diurnal variation of corticosterone levels, physiological and psychological stress, to which animals are subject during manipulation, may be the reason for a two-fold, or even higher, increase in serum glucocorticoid (39) and increased adrenal PBR levels. Indeed, it has been shown that PBR levels are under the control of both physiological and psychological stress (40, 41) suggesting that the receptor is involved in the stress response. Thus, in vivo treatment with EGb 761 may not affect the basal "unstressed" adrenal function but it may control the stress-induced PBR expression, thus maintaining lower "normal" circulating corticosterone levels. Since ACTH release is the most pronounced stress-response known, we considered that EGb 761 may not affect basal corticosteroid synthesis but it may control the response of adrenal cells to ACTH.

To examine this possibility we investigated the ex vivo effect of EGb 761 on adrenal steroidogenesis. For that, adrenocortical cells were isolated from rats treated in vivo with EGb 761, GKB, and saline and then incubated in culture with increasing concentrations of ACTH. In agreement with the in vivo data, treatment with EGb 761 resulted in a 50% reduction of corticosterone synthesized by the cells in response to ACTH, compared with cells isolated from saline-treated rats. Interestingly, using ACTH at concentrations lower than 100 pg/ml, no effect of EGb 761 could be seen on steroidogenesis. Considering that the physiological/circulating levels of ACTH are in the range of 20–50 pg/ml (30), these results demonstrate that EGb 761 does not affect normal adrenal function but only adrenal function under "stress" conditions, where ACTH levels are dramatically increased. A closer look at the ACTH dose-response curves indicate that in cells from extract-treated animals appear hyperbolic whereas cells from saline-treated animals showed the expected sigmoidal curve. This finding suggests that the process blocked by the drug treatment became rate-limiting for the cell response to ACTH. Thus, further receptor occupancy by ACTH will not produce any additional effect leading to higher steroid production.

The ex vivo effect of GKB treatment on ACTH-stimulated steroidogenesis was more pronounced (80% decrease), in agreement with its in vivo effect on circulating corticosterone levels (30). One should note, however, that despite the dramatic inhibition of ACTH-stimulated steroid synthesis by GKB, its effect became statistically significant only in the presence of 100 pg/ml or higher concentrations of ACTH. The ex vivo inhibitory effect of GKB on ACTH-stimulated steroid synthesis could not be seen after 12 days in culture. This finding is in agreement with our previous observation of the reversibility of the in vivo inhibitory effect of EGb 761 11 days after the end of the treatment. Because of the similarity of the responses to EGb 761 and GKB, we can conclude that the effects of EGb 761 are due to the GKB component in the extract. These studies provided us with the experimental paradigm to examine, under an ACTH-responsive and -unresponsive (or inhibited) state, the specific effects of treatment with EGb 761 and GKB and define elements involved in regulation of the hormone-stimulated steroid biosynthesis.

Incorporation of [35S]met/cys into proteins followed by 2D-PAGE analysis of the radiolabeled proteins has been extensively used for the identification of hormone-responsive polypeptides (15, 34, 42, 43). We used this approach to identify protein(s) targets of the ex vivo EGb 761 and GKB treatments in cultured adrenocortical cells. First, we confirmed our previous finding on the inhibition of PBR expression by EGb 761 and GKB. After 2 days in culture, adrenocortical cells isolated from saline-treated rats synthesized de novo a 17-kDa, pI 8 protein identified by 2D-PAGE immunoblot analysis as PBR. Treatment with EGb 761 and GKB inhibited ACTH-stimulated steroid synthesis and expression of this protein. More specifically, EGb 761 and GKB treatment reduced by 70% and 82% PBR protein expression, in agreement with our previous in vivo observations (30). It should be noted that the adrenal is one of the richest tissues in PBR, and even after a 70% reduction of PBR levels there is enough PBR remaining to sustain normal adrenal function in the presence of low, physiological, ACTH concentrations. After 12 days in culture, PBR protein synthesis recovered, whereas at the same time, the ex vivo GKB-induced inhibition of ACTH-stimulated corticosterone production ceased. Thus, the expression of PBR correlated with the ability of the cells to produce steroids in response to ACTH.

As we have previously observed and in the present studies under the conditions used for SDS-PAGE, the 18-kDa PBR isoquinoline-binding protein runs as a 17-kDa protein (8, 11). PBR is a multimeric complex (8, 44) where the isoquinoline-binding site is on the 18-kDa subunit, and expression of the benzodiazepine-binding site requires both the 18-kDa and the 34-kDa pore-forming voltage-dependent anion channel protein (8). Considering that voltage-dependent anion channel is located at the junctions between outer and inner mitochondrial membrane contact sites, these results suggest that the mitochondrial PBR complex could allow the translocation of cholesterol from the outer to the inner mitochondrial membrane. Further support for this hypothesis was provided by molecular modeling studies of the 18-kDa PBR protein (4, 45) and in vitro reconstitution studies in bacteria, where it was shown that PBR functions as a channel for cholesterol (4). Thus, the sum of the in vitro studies using mitochondria preparations (11, 14) and cells (10, 11, 13, 14) and of our previous in vivo (30) and the present ex vivo studies demonstrate that manipulation of PBR expression affects the ability of the cells to transport cholesterol, in response to hormone treatment, across the outer mitochondrial membrane and, thus, their competence to synthesize pregnenolone and the final steroid products. The cause-effect relation between the in vivo and ex vivo effects of EGb 761 and GKB on PBR expression and steroid synthesis is similar to that seen in steroidogenic cells devoid of PBR due to targeted disruption of the PBR gene (4). Thus, these results provide unequivocal evidence of the obligatory role of PBR in the regulation of steroid biosynthesis and validates the use of EGb 761 and GKB as pharmacological tools to modulate PBR levels.

In addition to PBR, two other proteins were identified as molecular targets of the EGb 761 and GKB treatments: a 40-kDa, pI 5.7–6 and a 32-kDa, pI 6.7 protein. The 40-kDa protein is unknown. However, we believe that the 32-kDa protein may be the precursor form of StAR (17).

In adrenocortical cells isolated from saline-treated rats and cultured for 2 days, we identified four proteins induced (40 kDa, pI 5.7–6; 29 kDa, pI 6.4–6.7; pI 32 kDa, 6.7; 90 kDa, pI 6.8–7.1) and one stimulated (85 kDa, pI 6.8) by ACTH. From these, the 29-kDa, pI 6.4–6.7 protein fits the characteristics of the ACTH-induced proteins in freshly isolated rat adrenocortical cells, described by Orme-Johnson and colleagues (15, 34). Proteins of similar molecular size and pI were shown to be induced by hormone treatment in various steroidogenic cells (34, 43, 46, 47). Stocco and colleagues found similar proteins (30 kDa) to be induced by hCG in MA-10 mouse tumor Leydig cells (42). In a series of studies these researchers demonstrated a close correlation between synthesis of the 30-kDa proteins and steroidogenesis (17). This 30-kDa protein was characterized and named StAR (17). It has been proposed that StAR is rapidly synthesized in response to hormone stimulation as a 37-kDa precursor protein. This protein is then targeted to mitochondria where, as it begins import and processing, contact sites of the outer and inner mitochondrial membranes are formed, and the signal sequence is removed, forming a 32-kDa form of the protein. Further processing of the protein at the mitochondrial level results in formation of the mature 30-kDa protein. It has also been proposed that during the processing of the protein and the accompanying formation of contact sites, cholesterol is transferred from the outer to the inner mitochondrial membrane, where it will be metabolized to pregnenolone.

The 29-kDa, pI 6.7 protein was recognized by an anti-StAR antiserum, raised against an internal peptide sequence of the MA-10 Leydig cell StAR protein (30), in 2D-PAGE immunoblot analysis. Interestingly, only the basic of the two forms of StAR was recognized by the antiserum, suggesting that posttranslational modification(s) of the most acidic form (i.e. phosphorylation) may be responsible for the loss of protein immunoreactivity. Our data show that the 29-kDa, pI 6.4–6.7 StAR protein is induced at the same level by ACTH in adrenocortical cells isolated from saline-, EGb 761-, and GKB-treated rats. Thus, its expression does not correlate with the inhibition of corticosteroid production. This finding is in agreement with our previous in vivo observation that treatment with GKB reduced circulating corticosterone levels, resulting in increased ACTH production and induction of StAR in the adrenal tissue (30). However, despite the stimulation of StAR expression, serum gluocorticoid levels remained low. In addition, adrenocortical cells maintained for 12 days in culture sustained their ability to synthesize steroids in response to ACTH, but not their capacity to synthesize the 29-kDa protein (StAR). Thus, these findings raise questions about the role of StAR (protein 3, pI 6.4–6.7, 29 kDa) as the sole mediator of the hormone-stimulated steroidogenesis.

The other proteins modulated by ACTH and/or EGb 761 and GKB treatment are not known. However, the fact that the expression of these proteins does not correlate with either the reversibility of the inhibition exerted by GKB or the ACTH-stimulated steroid synthesis makes them unlikely candidate mediators of the inhibitory effect of EGb 761 and GKB on hormone-dependent glucocorticoid formation.

Of interest is the finding that a 32-kDa, pI 6.7 protein is induced by ACTH in 2-day-old cultures of adrenal cells obtained from saline-treated rats only and not in cells from EGb 761- and GKB-treated rats. Thus, the expression of this protein in 2-day-old adrenocortical cell cultures correlates with changes in corticosterone synthesis. However, this protein is constitutively expressed by the 12-day-old adrenocortical cell cultures, suggesting that its presence may be either permissive or independent of the action of ACTH on steroidogenesis. PBR expression, on the other hand, is permissive for cholesterol transport and steroid biosynthesis. We previously showed that addition of hormones to steroidogenic cells results in changes in PBR topography and binding capacity, thus activating cholesterol transfer to P450scc and steroid production (48, 49). The molecular size and pI of this 32-kDa protein are remarkably similar to those of the StAR intermediary precursor protein (50, 51). In addition, the synthesis of both the 32-kDa protein and the StAR precursor is induced by hormones. Although additional studies are needed to define the identity of the 32-kDa protein, this finding may bring together the two main mechanisms described for cholesterol transport, PBR and StAR. Considering the recent findings that StAR protein may not need to enter the mitochondrion for activity (52), one could propose a model where the precursor StAR protein may mediate cholesterol transfer to the outer mitochondrial membrane or may mobilize steroidogenic cholesterol to specific areas of the outer membrane (contact sites?), whereas PBR mediates the transfer from the outer to the inner mitochondrial membrane. Such a model could explain the findings that mutations at the StAR gene are responsible for the expression of congenital lipoid adrenal hyperplasia disease (17), that steroid synthesis ceased in steroidogenic PBR-negative cell mutants (4), and that in vivo (30) and ex vivo inhibition of PBR expression reduces ACTH-stimulated glucocorticoid synthesis (present studies).

In conclusion, the G. biloba extract EGb 761 exerts its antistress or adaptive effects at least in part by regulating the levels of adrenal cortical PBR and two other target proteins, thus maintaining low levels of glucocorticoids and avoiding the stress-induced, long term, high glucocorticoid levels responsible for neurotoxicity and neuroendangerment (18, 19, 20, 53).


   Acknowledgments
 
We thank Dr. C. R. Jefcoate (University of Wisconsin, Madison, WI) for advising on the preparation of rat adrenal cortical cell cultures, and Dr. M. Culty (Georgetown University, Washington DC) for critically reviewing the manuscript.


   Footnotes
 
1 This work was supported by a grant from the Institut Henri Beaufour-IPSEN and Grant ES-07747 from the NIEHS, NIH (to V.P.). Back

2 Supported by Research Career Development Award HD-01031 from the NICHHD, NIH. Back

Received April 14, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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