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Endocrinology, doi:10.1210/en.2003-1669
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Endocrinology Vol. 145, No. 5 2165-2173
Copyright © 2004 by The Endocrine Society

Reversible Changes in Adrenocorticotropin (ACTH)-Induced Adrenocortical Steroidogenesis and Expression of the Peripheral-Type Benzodiazepine Receptor during the ACTH-Insensitive Period in Young Rats

J. J. Lee, P. Eisenberg, V. Papadopoulos, J. Wang and E. P. Widmaier

Department of Biology (J.J.L., J.W., P.E., E.P.W.), Boston University, Boston, Massachusetts 02215; and Department of Biochemistry and Molecular Biology (V.P.), Georgetown University School of Medicine, Washington, D.C. 20007

Address all correspondence and requests for reprints to: Dr. E. P. Widmaier, Department of Biology, Boston University, 5 Cummington Street, Boston, Massachusetts 02215. E-mail: widmaier{at}bu.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We previously demonstrated decreased adrenal content of a mitochondrial cholesterol transporter [peripheral-type benzodiazepine receptor (PBR)] during the first postnatal week in rats, when ACTH-inducible steroidogenesis is low. Here we report that the expression of PBR protein and mRNA increases throughout the neonatal/juvenile period in rats in parallel with ACTH-inducible steroidogenesis in vitro. We also previously reported that chronic stimulation of rat pups with daily ACTH injections augmented the steroidogenic response of the developing adrenal cortex. We therefore tested the hypotheses that the change in phenotype induced by ACTH was permanent, and that the effects of ACTH were mediated by increased PBR expression. Pups were injected with ACTH or saline from postnatal d (pd) 2–8 or 2–14. Another group of pups received ACTH from pd 2–7, followed by saline from pd 8–14. On the final day, all pups were challenged with a test injection of ACTH or saline. Exposure to ACTH, but not saline, for 1 wk significantly increased adrenal mass, the corticosterone response to ACTH, and the expression of PBR protein and mRNA. Continued ACTH treatment for a second week maintained adrenal mass, steroidogenesis, and PBR mRNA expression. When ACTH was withdrawn after 1 wk and replaced with daily saline injections, however, adrenal mass, ACTH-inducible steroidogenesis, and PBR expression returned to levels comparable to those in age-matched saline controls (i.e. animals that had not received ACTH injections during the first 2 wk). Thus, although ACTH was capable of inducing increased steroidogenic capacity of the juvenile rat adrenal, its effects were only manifest when pups were exposed regularly to high plasma ACTH levels. ACTH, therefore, has significant, but reversible, effects on the development of adrenocortical function, possibly mediated in part by increased expression of PBR.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
MAMMALS BORN BEFORE fetal adrenocortical cells gain the ability to synthesize glucocorticoids exhibit a host of life-threatening pathologies that can be prevented or ameliorated with prenatal glucocorticoid therapy. Because of the importance of functional adrenal glands for postnatal survival and tissue and organ differentiation (1), it is critical that an understanding be achieved of the mechanisms by which adrenal glands differentiate from an immature into a mature phenotype. One functional marker of phenotypic maturation is the development of ACTH sensitivity, whereby adrenocortical cells acquire the capacity to respond to ACTH with high rates of glucocorticoid synthesis. The timing of the onset of adrenocortical sensitivity to ACTH stimulation, therefore, is a key event in fetal and postnatal life. The mechanisms that govern the onset of maximal adrenocortical sensitivity to ACTH stimulation, however, are uncertain, although it is clear that the presence or absence of key transcriptional activators and inhibitors are required for organogenesis and steroidogenesis (2, 3, 4).

We have previously reported that ACTH-inducible steroidogenesis both in vitro and in vivo is significantly lower in neonatal rats than in adults (5, 6). We also reported that the expression and activities of microsomal enzymes involved in the conversion of cholesterol to glucocorticoids, particularly 3ß-hydroxysteroid dehydrogenase, are low during the postnatal period (7). Similarly, the expression of 3ß-hydroxysteroid dehydrogenase mRNA is low in human fetal adrenal glands and high in adult glands (8). These results suggested that steps within the biosynthetic pathway from cholesterol to glucocorticoids were key determinants in the ability of developing adrenocortical cells to assume a mature phenotype. In addition, we demonstrated that at very early ages after birth, the expression and activity of adrenal peripheral-type benzodiazepine receptor (PBR) protein, a mitochondrial protein required for the transport of cholesterol across the outer mitochondrial membrane to its initial site of enzymatic cleavage (9, 10), was significantly lower than that in adult adrenal glands (6). However, that study did not characterize when during development PBR protein increased to levels comparable to those seen in adult adrenals, nor was PBR mRNA examined. Notwithstanding, these studies and a recent report demonstrating age-dependent increases in the expression of the ACTH receptor (MC2R) in rat adrenals (11) suggest that there are multiple loci at which the steroidogenic response to ACTH is developmentally regulated in rodents; this is further supported by studies in fetal sheep (12).

We reported that injections of ACTH (but not glucocorticoids) at physiological, stress-like levels into newborn rat pups for 1 wk resulted in increased sensitivity to a subsequent ACTH challenge, which could be mimicked by daily exposure of pups to ether stress (13). Those results suggested to us that long-term exposure of rat adrenal cells to ACTH in vivo may accelerate the normal maturational processes within the gland that lead to a functionally mature phenotype. If true, we would predict that the phenotype would be maintained even when circulating ACTH concentrations were allowed to return to normal after the treatment period. Moreover, because PBR is required for steroidogenesis (9, 10), and its expression is low during the first week of postnatal life in rats (6), we predicted that chronic ACTH injections would also increase the expression of PBR, and that the expression would persist even after the ACTH injections were discontinued, thus producing a permanently altered phenotype.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
For all experiments in this and subsequent studies (below), timed pregnant Sprague Dawley rats (Harlan, Indianapolis, IN) were obtained on gestation d 18 and housed singly with food and water freely available (lights on 0700–1900 h). The day of birth was designated postnatal d (pd) 1. To compare the developmental profiles of PBR protein and mRNA expression with that of ACTH-inducible steroidogenesis in vitro, adult rats or pups (various ages between pd 1–38) were decapitated between 0800–1000 h. Because there are no sex differences in PBR expression in rat adrenals (14, 15), animals of both sexes were used for all experiments (adults and pups).

Developmental profile of ACTH-induced steroidogenesis
Adrenal glands were removed, decapsulated to remove most of the outer connective tissue and glomerulosa cell layer, and pooled for a given age group. The pooled tissue was used for acute cell isolation and analysis of in vitro sensitivity to ACTH. Briefly, the tissue was subjected to enzymatic digestion as previously described (5, 6, 16). Approximately 105 viable adrenocortical cells (identified as those cells containing large vacuoles consistent with the appearance of lipid droplets and excluding the dye trypan blue) were incubated in 1 ml incubation medium [DMEM/Ham’s F-12 plus 0.1% (wt/vol) BSA] with or without 1 ng/ml porcine ACTH1–39 (Sigma-Aldrich Corp., St. Louis, MO) for 2 h at 37 C in a humidified 95% O2/5% CO2 atmosphere. At the end of the incubation, the suspensions were centrifuged, and the supernatants were collected for RIA of corticosterone (ICN Pharmaceuticals, Costa Mesa, CA). At the youngest ages, the efficiency of decapsulation may be less than at later ages, and it is possible that different amounts of underlying fasciculata cells could be removed by this procedure at very young ages. However, all data were normalized not to adrenal mass, but to number of viable cortical cells as described above, thereby minimizing any potential impact of the decapsulation procedure.

Preparation of whole cell homogenates and Western blot analysis
Whole cell homogenates were prepared by collecting decapsulated adrenals from adult rats (both sexes) and pups (both sexes) of various ages in homogenization buffer (50 mM Tris/0.25 M sucrose, pH 7.4) on ice. The numbers of adrenal glands pooled for pd 1–10, pd 15–20, pd 25–39, and adult rats were 40–55, 25–35, 4–6, and 3–4, respectively. For injection studies (see below), the numbers of adrenal glands pooled for pd 8 and pd 14 pups were 8–15 and 15–18. Tissues were washed once with buffer and then homogenized with a Tissuemizer (Tekmar, Cincinnati, OH) at 6000 rpm for 45 sec. Protein concentrations were quantified by Bradford assay (Bio-Rad Laboratories, Hercules, CA) with BSA as standard. Equal amounts of protein (15 µg) in each age group were fractionated by one-dimensional SDS-PAGE on 15% polyacrylamide gels. Proteins were then transferred onto polyvinylidene difluoride membranes (Bio-Rad Laboratories) and blocked in Tris-buffered saline/Tween 20 (TBST) containing 3% (wt/vol) nonfat milk for 1 h at room temperature. Membranes were then incubated with primary antiserum to PBR (1:2000) in blocking buffer overnight at 4 C as previously described (6, 17, 18). The blots were washed three times in TBST and incubated with secondary IgG conjugated to horseradish peroxidase (1:5000; Sigma-Aldrich Corp.). After exposure to film, blots were washed four times in TBST, then incubated in stripping buffer [62.5 mM Tris-HCl (pH 6.8), 2% SDS, and 100 mM ß-mercaptoethanol] for 30 min at 50 C. Membranes were washed six times in TBST, then blocked in buffer and reprobed with monoclonal ß-actin antibody (1:1000; Sigma-Aldrich Corp.). Membranes were then stripped a second time as described above and reprobed for the processed form of steroidogenic acute regulatory (StAR) protein as previously described (6). Immunoreactive proteins were visualized by chemiluminescence using the ECL detection reagent (DuPont-NEN Life Science Products, Boston, MA). Densitometric analysis of the immunoreactive protein bands was performed using the ImageJ 1.30 program (NIH, Bethesda, MD).

Northern blot analysis
Total RNA from adrenal tissue collected as described above was isolated using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA). Total RNA concentrations were determined by measuring absorbency of UV light at 260 nm. Oligonucleotide probes for PBR, steroidogenic factor-1 (SF-1), dosage-sensitive sex reversal adrenal hypoplasia congenita, critical region on the X chromosome gene-1 (DAX-1), and ß-actin were designed based on rat cDNA sequences from GenBank using PrimerQuest software (Integrated DNA Technologies, Coralville, IA). Custom oligonucleotide probes (Invitrogen Life Technologies) were 5' end labeled with [{gamma}-32P]ATP using the KinaseMax 5'-End-Labeling Kit (Ambion, Inc., Austin, TX). RNA was size-fractionated by electrophoresis and transferred to positively charged nylon membranes (Roche, Indianapolis, IN). Membranes were prehybridized for 30 min at 45–50 C in ULTRAhyb-Oligo solution (Ambion, Inc.), then hybridized overnight with labeled probes. After hybridization, membranes were washed once in 2x sodium chloride/sodium citrate and 0.1% sodium dodecyl sulfate at room temperature, followed by two washes under high stringency conditions (0.2x sodium chloride/sodium citrate and 0.1% sodium dodecyl sulfate at hybridization temperature). Membranes were exposed to Bio-Max film (Eastman Kodak Co., Rochester, NY) overnight at –80 C. To control for RNA loading variability, membranes were stripped and rehybridized with 32P-labeled rat ß-actin oligonucleotide probe. Densitometric analysis of the total RNA expression bands was performed using the ImageJ 1.30 program (NIH).

RNA analysis by RT-PCR
The developmental patterns of mRNA expression of SF-1 and DAX-1 as well as the endogenous ligand for PBR known as diazepam binding inhibitor (DBI) and the coactivator of PBR known as the PBR- and PKA-associated protein-7 (PAP-7) were also assessed in part by semiquantitative RT-PCR using Promega reagents. Total RNA was isolated as described above, and 18S RNA served as a control in each amplification reaction. cDNA was created using 2.5 ng RNA. All primers were designed based on cDNA sequences in GenBank using PrimerQuest software (Integrated DNA Technologies), and are given in Table 1Go. Pilot studies were performed to optimize PCR conditions that resulted in exponential amplification of product. Maximal differences for SF-1, DAX-1, and 18S were obtained between cycles 25 and 30. For DBI and PAP-7, the optimal cycle was between 30 and 35. Amplification products were separated on a 1.5% agarose gel and stained with ethidium bromide. Resulting bands of the PCR products were quantified using the ImageJ 1.30 program (NIH). The identities of all PCR products were confirmed by extracting the bands from the gel and sequencing the products using the ABI PRISM big dye terminator sequencing kit and the 377 Automated DNA Sequencer (PE Applied Biosystems, Foster City, CA).


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  		TABLE 1. Primers used in RT-PCR experiments

 
Effects of chronic ACTH injections on ACTH-inducible steroidogenesis and PBR expression
To determine whether chronic exposure to ACTH in vivo would up-regulate steroidogenesis and PBR expression, dams and their litters were randomly assigned to one of several treatment groups. Each treatment group was represented by individuals within each litter. Pups of both sexes were injected ip twice daily (0800 and 1600 h) beginning on pd 2 with either saline or 20 µg/kg porcine ACTH1–39 in a volume of 100 µl. On pd 8, half of the pups from each group were randomly divided into two additional groups receiving a final injection of either saline or ACTH, then killed by decapitation 30 min later for collection of blood and tissue samples. The remaining pups were injected for an additional 6 d with either saline or ACTH, then challenged on the final day (pd14) with either saline or ACTH as described above. One group of pups was left untreated throughout the experiment and killed on the last day. Expression of PBR and StAR protein and PBR mRNA transcript was determined as described above. Because hormone, adrenal mass, and PBR expression were not significantly different between this group and saline-treated controls, the two groups were pooled. In some experiments, ACTH injections were performed from pd 3–10 without a second week of injections. No significant differences in any measured variable was observed between groups injected from pd 2–8 or pd 3–10, and thus data from these animals were pooled. Blood was collected into heparin-free, EDTA-containing tubes and centrifuged for RIA of plasma ACTH and corticosterone (ICN Pharmaceuticals). The left adrenal gland from each animal was removed and weighed. In one experiment, individual animal body masses were obtained to normalize adrenal mass. To minimize time and handling required to perform the experiments (to minimize nonspecific stressors), however, in subsequent experiments a single average body mass was obtained en masse for each treatment group, which was used to normalize adrenal mass. All experimental procedures were approved by the Boston University institute animal care and use committee.

Data analysis
ACTH, corticosterone, and adrenal mass data were analyzed by one-way ANOVA, followed by relevant comparisons with Bonferroni correction. Age-dependent expressions of RNA transcripts were compared relative to adult values in the case of PBR and relative to pd 1 values for DAX-1 and SF-1 (in which expression decreased or did not change with age, unlike that for PBR, which increased with age). For statistical analyses, these data were arcsin-transformed before ANOVA and post hoc tests, with significance set at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Corticosterone production from isolated rat adrenocortical cells in vitro significantly increased with age of the pups, as expected (Fig. 1Go)



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  		FIG. 1. Stimulation of corticosterone production from adrenal cells in vitro during postnatal ages and in adulthood. Acutely isolated rat adrenal cells from decapsulated adrenal glands were incubated with or without 1 ng/ml porcine ACTH1–39 for 2 h, at which time the cell suspension was centrifuged, and the supernatant was removed for corticosterone RIA. Results are expressed as the fold increase in corticosterone produced after ACTH stimulation compared with vehicle controls. Each point represents the mean and SE of two to four separate experiments. Basal levels of corticosterone production ranged from 8–116 ng/106cells·h.

 
Expression of immunoreactive PBR (irPBR) protein in whole cell homogenates significantly increased with age, beginning slightly earlier than the increase in ACTH-inducible steroidogenesis (Fig. 2Go, top panel). Similar results were found when PBR protein levels were quantified in purified mitochondria (not shown). In one preliminary experiment, no change in the expression of mRNA transcripts of PBR-associated proteins (PAP-7 and DBI) was observed across ages as assessed by RT-PCR (not shown), nor was there any significant age-dependent change in the expression of immunoreactive StAR protein in three experiments (Fig. 2Go, lower panel). Therefore, the developmental patterns of expression of these three proteins were not further examined. The increased expression of PBR protein was the result of increased synthesis, because it was associated with increased, age-dependent expression of PBR mRNA (Fig. 3Go).



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  		FIG. 2. Changes in irPBR expression (top panel) or StAR expression (lower panel) in whole homogenates of decapsulated rat adrenal glands during the postnatal period (age in postnatal days given above each lane) and adulthood. A representative Western blot with irß-actin (loading control) is shown in the inset. Data from multiple separate experiments were summarized and analyzed by comparing the PBR/ß-actin or StAR/ß-actin ratios at each age to the ratio in adults, which was set at 1.0. Each point represents the mean and SE of two (pd 35 and 39) or five (all others) experiments. There was an overall significant increase in PBR expression by ANOVA; all values at ages less than pd 39 were significantly lower than adult values (at least P < 0.05). There was no overall significant effect of age on StAR expression.

 


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  		FIG. 3. Changes in PBR mRNA expression in decapsulated rat adrenal glands (PBR/ß-actin ratios expressed relative to adults). Total RNA was extracted from pooled adrenal glands at each age (postnatal days indicated above each lane), and subjected to Northern blot analysis using oligonucleotide probes as described in Materials and Methods. A representative blot is shown in the inset, and the summarized data (mean and SE) of three separate experiments are shown in the graph. There was an overall significant increase in the expression of PBR mRNA by ANOVA; all values at ages less than pd 25 were significantly lower than adult values (at least P < 0.05).

 
No significant change was observed in the expression of the orphan nuclear receptor SF-1 during the neonatal/juvenile period (Fig. 4AGo). The expression of the putative inhibitor of SF-1 action, DAX-1, significantly decreased at later juvenile ages (Fig. 4BGo). Although the data in Fig. 4Go are derived from RT-PCR and are therefore only semiquantitative, the results were verified in one experiment by Northern blot analysis. In that case, no change was observed in SF-1 expression when normalized to ß-actin expression, but DAX-1 expression decreased from pd35 onward (Fig. 4CGo).



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  		FIG. 4. SF-1 and DAX-1 mRNA expression in decapsulated adrenal glands from rat pups (postnatal ages indicated above each lane) and adult rats (SF-1 or DAX-1/18S ratio expressed relative to the value on pd 1). A representative gel is shown for each in the insets; summarized data from three separate experiments are shown in the graphs (mean and SE). There was no overall significant change in SF-1 expression (A). For DAX-1 (B), all values at ages younger than pd 35 were significantly greater than adult values. C, Northern blot analysis of DAX-1, SF-1, and ß-actin at the ages shown in A and B.

 
Injections of ACTH resulted in significant increases in plasma ACTH 30 min after injection in all groups receiving the injections regardless of prior treatment history, as expected (Fig. 5Go, top panel). Plasma ACTH levels were increased to levels approximately 1.5–2.0 times greater than those observed in stressed neonatal or juvenile rats (19, 20).



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  		FIG. 5. The effects of repeated stimulation with ACTH on the appearance of an adult-like adrenal phenotype (increased sensitivity to ACTH). Postnatal d 2 rat pups were injected twice daily with chronic saline (S) or ACTH (A) for either 1 or 2 wk; in some cases, animals received ACTH injections for the first week and then saline for the second week. Rats were then challenged with a final acute injection of saline (S) or ACTH (A) and were killed 30 min later for determination of plasma ACTH (top panel) and corticosterone (lower panel). A dashed line indicates that the animals were killed after the first week. Values are the mean ± SE. *, At least P < 0.05 vs. each other. NS, Not significantly different from each other. Only relevant pairwise comparisons are indicated. Plasma ACTH was determined from a random subset of animals (n = 44–50 for wk 1; n = 26–27 for wk 2). For plasma corticosterone, n = 99–104 for wk 1 and 33–37 for wk 2.

 
The steroidogenic response to ACTH injection on pd 8 was significantly greater in animals that had been previously exposed to daily ACTH injections (Fig. 5Go, lower panel), consistent with our earlier report (13). However, when ACTH injections were discontinued after 1 wk and replaced with daily saline injections, the up-regulation of ACTH-inducible steroidogenesis was no longer present on pd 14, and the corticosterone response was similar to that observed in pd 8 pups treated for the first time with ACTH. By contrast, when ACTH injections were continued from pd 8–14, the increased steroidogenic response to a final ACTH challenge remained significantly greater than in saline-treated, age-matched controls (Fig. 5Go, lower panel).

Acute ACTH injections had no significant effect on adrenal mass, and thus the data for the acute treatment groups (saline or ACTH final injection) were pooled for analyses. The effects of ACTH on adrenal mass were observed whether individual body mass measurements were obtained for each pup or if average body masses were obtained en masse as described in Materials and Methods. Repeated ACTH injections significantly increased left adrenal mass compared with saline-injected controls (Fig. 6Go). The effect was significant by pd 8 and remained significant after an additional week of ACTH injections. As with the increased steroidogenic response, when ACTH injections were discontinued after 1 wk, adrenal mass did not continue to increase and was not different from that in age-matched saline controls on pd 14. Only in the presence of continued exposure to ACTH was adrenal mass increased above age-matched control values on pd 14 (Fig. 6Go).



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  		FIG. 6. The effect of ACTH (A) or saline (S) for either 1 or 2 wk on adrenal mass in juvenile rat pups. Acute challenge with ACTH had no effect on mass, and thus the final treatment groups (acute injections of saline or ACTH) are combined. Adrenal mass was significantly increased compared with controls on pd 14 only when ACTH injections were continued during the second treatment week. Values are the mean and SE of 45–79 animals. *, At least P < 0.05 vs. each other. NS, Not significantly different from each other. Only relevant pairwise comparisons are indicated.

 
In one experiment, animals from each injection group (saline or ACTH from pd 2–8; saline or ACTH from pd 2–14 or ACTH from pd 2–8, followed by saline from pd 9–14) were killed, and the adrenals were collected to directly confirm that in vitro sensitivity to ACTH was increased. Cells were incubated as described in Fig. 1Go (except that the incubation period was only 30 min). In that experiment, ACTH induced a 6-fold increase in corticosterone secretion in vitro in cells from animals previously treated with saline injections on pd 2–8, and a 23-fold increase in secretion in cells obtained from animals treated daily with ACTH on pd 2–8. On pd 14, ACTH induced a 2-fold or less increase in corticosterone secretion in cells from animals previously treated with either saline for 2 wk or ACTH for 1 wk, followed by saline for a second week. In animals treated with ACTH throughout pd 2–14, however, adrenal cells responded to ACTH stimulation with a 22-fold increase in corticosterone secretion (not shown). Thus, the effects of ACTH in vivo (Fig. 5Go) were also observed in adrenal cells incubated directly with ACTH in vitro after the animals had been previously treated with ACTH injections in vivo.

Because the expression of PBR protein and transcript closely paralleled that of ACTH-inducible steroidogenesis in developing rat adrenal glands (Figs. 1–3GoGoGo), we examined both protein and mRNA levels for this important mediator of cholesterol transport into the mitochondria after exposure to ACTH. As predicted from Fig. 2Go, irPBR expression slightly increased between pd 8 and pd 14 (Fig. 7Go, top panel). Consistent with our previous report (6), there was no significant acute effect of ACTH on PBR expression. Thus, the data for each pair of final treatments (saline or ACTH challenge test on final day) were pooled for analyses, as shown in the graphs of Figs. 7Go and 8Go. Chronic ACTH injections increased the expression of irPBR (Fig. 7Go, top panel), but had no significant effect on irStAR protein (Fig. 7Go, lower panel). In addition, chronic ACTH injections significantly increased PBR mRNA levels (Fig. 8Go) during the first treatment week. One week after discontinuation of ACTH injections, however, when ACTH-induced steroidogenesis had returned to levels in age-matched saline controls, the expression of PBR mRNA was no longer different between animals that had received ACTH and animals that received saline injections during the first treatment week (Fig. 8Go). By contrast, when ACTH injections were continued during a second week, PBR mRNA levels (like ACTH-induced steroidogenesis) remained significantly greater than in animals injected only with saline for both weeks or with ACTH for only 1 wk, followed by saline the second week (Fig. 8Go). A similar trend was observed with irPBR protein during the second week of treatment (Fig. 7Go, top panel), but differences on pd 14 did not reach statistical significance, possibly due to relatively high variability between blots. No significant changes were observed in the expression level of mRNA transcripts for DAX-1 or SF-1 (not shown).



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  		FIG. 7. Effect of chronic ACTH injections on the expression of irPBR (top panel) or irStAR (lower panel) in whole cell homogenates of decapsulated rat adrenal glands. Nine to 15 adrenals from individual pups were pooled for each treatment group. Injection protocols were the same as those in Fig. 5Go (A, ACTH; S, saline). All blots, including the representative blots, were probed with anti-PBR antiserum, stripped, and reprobed with anti-ß-actin antibodies and then anti-StAR antiserum. Acute ACTH injections did not significantly alter PBR or StAR protein expression. Therefore, the various treatment groups shown in the blots were combined according to the type of injections received during wk 1 and 2. The PBR or StAR/actin ratio from each group was normalized to a no treatment control group that was included in each experiment and that was not significantly different from the saline control group (not shown). The data represent the mean ± SE of three separate experiments. *, At least P < 0.05 vs. each other.

 


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  		FIG. 8. Effect of chronic injections of ACTH on the expression of PBR mRNA in decapsulated rat adrenal glands. Total RNA was extracted from pooled adrenal glands from each treatment group and subjected to Northern blot analysis using oligonucleotide probes as described in Materials and Methods. The inset shows a representative Northern blot for PBR and ß-actin mRNA. Injection protocols were as described in Figs. 5–7GoGoGo. Acute ACTH injections did not significantly affect PBR mRNA expression. Therefore, the various treatment groups were combined according to the type of injections received during wk 1 and 2 as described in Fig. 7Go. The PBR/actin ratio from each group was normalized to a no treatment control group that was included in each experiment and which was not significantly different from the saline control group (not shown). The data represent the mean ± SE of three separate experiments. *, At least P < 0.05 vs. each other.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The major findings in this study were that the ontogenic patterns of PBR protein and mRNA expression and ACTH-inducible steroidogenesis in young rat adrenal glands in vitro were highly similar. Both PBR expression and ACTH-inducible steroidogenesis were up-regulated by chronic exposure of pups to ACTH in vivo, and both variables returned to age-matched control levels when ACTH exposure was discontinued. No other examined factor associated with steroidogenesis or adrenocortical development covaried with ACTH exposure or with age in this way.

The ability of the mammalian adrenal gland to synthesize large amounts of glucocorticoids typically does not occur until fairly late in gestation, as in sheep (12, 21) and humans (21), or even postnatally, as in rats (22) and mice (23). It has been generally considered that exposure of the developing fetus or neonate to chronic high levels of glucocorticoids may be detrimental to the developing brain and other organs (22, 24, 25). By contrast, basal levels of glucocorticoids are clearly important for the growth and development of numerous fetal and neonatal tissues, including the lungs (26) and brain (24). Thus, a balance must be achieved between maintenance levels of adrenal steroids that facilitate tissue differentiation and stress levels of steroids that could be toxic to the same or other tissues. In this context, we have previously demonstrated that baseline (unstimulated) corticosterone production in vitro is greater in adrenal cells of neonatal rat pups than in adult cells, where it is nearly negligible (5). Conversely, ACTH-inducible corticosterone production is much greater in adult cells than in cells from neonatal rat adrenals (5, 27), although before this report we have not documented precisely when maximal ACTH sensitivity is first achieved. The results of the present study demonstrate that this does not occur until fairly late in the juvenile period. However, we previously reported that in vivo plasma corticosterone response to insulin-induced hypoglycemia stress was roughly similar in pd 19 pups and adult rats (20). The disparity between the in vivo and in vitro results could be explained by the presence of additional stress-induced factors besides ACTH that may act in concert with the peptide to further stimulate adrenal activity in vivo. In addition, although we have no evidence to support it, there remains the possibility that adrenal cells of rats of different ages may be differentially sensitive to the effects of acute cell isolation.

The cellular mechanisms that determine when and how an immature adrenocortical cell attains maximal ACTH sensitivity, however, are only partially understood. Age-dependent changes have been documented in microsomal enzymes involved in the conversion of cholesterol metabolites to glucocorticoids (7), in the expression of ACTH receptors and adenylate cyclase activity in the membranes of adrenal cells of fetal sheep (12) or postnatal rats (11), and in the expression of two mediators of cholesterol transport to mitochondria (PBR and StAR) (6, 28). We previously determined that adrenal PBR protein levels and binding activity were significantly lower in the early postnatal stages in rats compared with adult adrenals, but in that study we did not examine PBR mRNA levels or determine when PBR expression increased to adult-like levels (6). In the present study we found that the expression of PBR (both protein and mRNA) significantly increased during the course of the neonatal and juvenile periods. There was an apparent step increase in PBR protein expression between pd 10–15, the basis of which we do not yet understand. However, the increase in protein expression observed at that time correlates with a small, but steady, increase in PBR ligand-binding activity around those ages that we reported previously (6).

The pattern of expression of both PBR protein and its transcript closely paralleled that of the development of ACTH-inducible corticosterone production. Similar changes in the expression of two proteins, believed to facilitate maximal PBR protein activity (PAP-7 and DBI), and in StAR protein were not observed postnatally. Although the data are correlational and do not prove that the increase in PBR expression is responsible for the increase in the functional maturation of rat adrenocortical cells, the close and specific relationship is suggestive of a link between the two events, particularly as it is clear that PBR expression is an absolute requirement for adrenal steroidogenesis (9, 29).

In support of the hypothesis that the developmentally timed increases in PBR expression and ACTH-inducible steroidogenesis are functionally linked, however, was the observation that chronic exposure of pups to ACTH injections increased both PBR protein and mRNA levels and also increased the ability of pups to respond to a subsequent ACTH challenge with significantly increased corticosterone production. It should be noted that the plasma levels of ACTH achieved after injection were consistent with what would be high stress levels in young rats. We previously reported that similar effects of ACTH on steroidogenesis could be obtained with much lower levels of plasma ACTH (13), but we do not yet know whether similarly low ACTH levels would also be effective in up-regulating PBR expression in parallel.

The increased plasma levels of corticosterone after ACTH challenge in animals that were chronically exposed to ACTH in vivo, most likely reflects increased steroidogenesis (as opposed to changes in steroid half-life in plasma), as in one experiment adrenal cells from animals treated in vivo for 1 or 2 wk with ACTH showed greater ACTH-induced corticosterone production in vitro than cells obtained from animals treated in vivo with saline. Of interest is that chronic ACTH exposure did not result in elevated basal corticosterone levels in vivo despite the increase in relative adrenal mass. This observation is consistent with our earlier suggestions that basal steroidogenesis in the neonatal and juvenile periods in rats is at least partly ACTH independent (5, 6). Further support of the hypothesis that PBR expression and ACTH-inducible steroidogenesis are linked during the neonatal/juvenile period is the recent observation that exposure of newborn rats to chronic hypoxia stress results in both increased ACTH-induced adrenal steroidogenesis and increased adrenal PBR expression (30).

Not only did ACTH injections result in increased PBR expression after 1 wk, but when ACTH injections were discontinued for another week, both ACTH-induced steroidogenesis and PBR expression were no longer different from those in age-matched animals that had never received ACTH injections. By contrast, continued exposure to ACTH maintained significantly greater PBR mRNA expression and ACTH-inducible steroidogenesis and adrenal mass. Thus, the effect of ACTH on the phenotype of the developing rat adrenal was reversible and not permanent, in contrast to our original hypothesis. Moreover, PBR expression and ACTH-inducible steroidogenesis covaried during the course of postnatal life, after either chronic ACTH exposure or after removal of the ACTH stimulus. No other intracellular mediator of steroidogenesis that was tested in this study varied in this way, including the orphan nuclear receptors SF-1 and DAX-1, which are known to be important in adrenal organogenesis and stress responses (2, 3, 4). The expression of StAR protein was also unchanged. This does not, however, rule out a possible role for StAR in the changes in adrenal sensitivity to ACTH induced by chronic exposure to ACTH, as we did not examine multiple time points after ACTH injection. In addition, it should be noted that we recently reported that StAR (and PBR) may be involved in the increased adrenal response to chronic hypoxia observed in young rats (30).

Basal expression of DAX-1, however, significantly decreased at late juvenile ages, consistent with a recent report (31). DAX-1 appears to act as a repressor of the steroidogenic actions of SF-1 (2, 4) and is required for normal adrenal organogenesis (32). Therefore, we hypothesized that DAX-1 gene expression would decrease in rat adrenal glands around the time that the glands became sensitive to the steroidogenic effects of ACTH. Our data only partially support this hypothesis, because the method used to assess DAX-1 expression was not highly quantitative, and the temporal relationship between the onset of ACTH-inducible steroid production and the decrease in DAX-1 expression was not nearly as close as that between PBR and steroidogenesis. Nonetheless, it is possible that a decrease in the expression of DAX-1 plays a role in the final stages of the maturation of the adrenal response to ACTH.

We suggest, therefore, that the specific and covarying changes in PBR expression and ACTH-inducible steroidogenesis may indicate that PBR is a key mediator of ontogenic changes in adrenal sensitivity to ACTH. Short-term stress (maternal deprivation) has been shown to increase adrenal sensitivity to ACTH in vivo within 24 h (33). It would be of interest to determine whether adrenal PBR expression is affected by ACTH treatment in young rats in a similar timeframe. Notwithstanding, it is highly likely that a coordinated developmental sequence of PBR, DAX-1, and plasma and intracellular membrane components of the steroidogenic process must occur in tandem for the mammalian adrenal cortex to switch from an ACTH-insensitive to a fully ACTH-responsive state. Moreover, it is also clear that the effects of ACTH on adrenal sensitivity to ACTH are not permanently imprinted on the newborn/juvenile rat adrenal.


   Acknowledgments
 
We thank Mr. Christopher Wall for technical assistance.


   Footnotes
 
This work was supported by National Institutes of Health Grant DK-55793 (to E.P.W.) and in part by the Undergraduate Research Opportunities Program at Boston University (to P.E.).

Abbreviations: DAX-1, Dosage-sensitive sex reversal adrenal hypoplasia congenita, critical region on the X chromosome gene-1; DBI, diazepam binding inhibitor; ir, immunoreactive; PBR, peripheral-type benzodiazepine receptor; SF-1, steroidogenic factor-1; StAR, steroidogenic acute regulatory; TBST, Tris-buffered saline/Tween 20.

Received December 9, 2003.

Accepted for publication February 4, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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