Endocrinology Vol. 140, No. 2 859-864
Copyright © 1999 by The Endocrine Society
Developmental Expression of the Peripheral-Type Benzodiazepine Receptor and the Advent of Steroidogenesis in Rat Adrenal Glands1
Alexandra Zilz,
Hua Li,
Rosa Castello,
Vassilios Papadopoulos and
Eric P. Widmaier
Department of Biology (A.Z., R.C., E.P.W.), Boston University,
Boston, Massachusetts 02215; and Departments of Cell Biology (V.P.,
H.L.) and Pharmacology (V.P.), Georgetown University Medical Center,
Washington, D.C. 20007
Address all correspondence and requests for reprints to: Dr. Eric P. Widmaier, Department of Biology, 5 Cummington Street, Boston University, Boston, Massachusetts 02215. E-mail:
widmaier{at}bio.bu.edu
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Abstract
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Although the precise mechanism whereby cholesterol is transported
across the outer mitochondrial membrane is uncertain, a multimeric
receptor complex termed the peripheral-type benzodiazepine receptor
(PBR) appears essential for this process. We therefore predicted that
adrenal cells at different developmental stages would express PBR
coincidentally with the advent of steroidogenesis. Adrenals of neonatal
rats demonstrate greatly reduced sensitivity to ACTH that gradually
increases after the first 2 weeks of life. Thus, neonates have lower
circulating corticosterone levels following exposure to stress. We
examined mitochondrial PBR ligand binding activity, immunoreactive (ir)
PBR content, and adrenal sensitivity to ACTH in vivo and
in vitro. Ontogeny of both mitochondrial PBR ligand
binding capacity and irPBR directly paralleled that of ACTH-inducible
steroidogenesis in isolated rat adrenal cells and in rats injected with
ACTH. In addition, neonatal PBR had approximately 2-fold higher
affinity for PK11195, a synthetic ligand that binds with high affinity
to PBR. No correlation was observed during neonatal life between
ir-steroidogenic acute regulatory (StAR) protein content and
steroidogenesis. These results are consistent with the hypothesis that
PBR is an absolute prerequisite for adrenocortical steroidogenesis, and
suggest that the stress hyporesponsive period of neonatal rats may
result from decreased PBR expression. In addition, the higher affinity
of neonatal PBR and the relatively high basal expression of StAR
protein in neonatal adrenals may partly explain the high constitutive
steroidogenesis characteristic of neonatal rat adrenal cells.
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Introduction
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THE RATE-LIMITING step in steroidogenesis
is transport of free cholesterol from intracellular stores to the inner
mitochondrial membrane, where the first steroid biosynthetic enzyme is
located (cytochrome P450scc). The process whereby cholesterol is
generated and targeted to the mitochondria begins with the generation
of cAMP following binding of ACTH to its membrane receptor. The precise
mechanism whereby cholesterol reaches the outer mitochondrial membrane,
and is then transported to the inner membrane at contact points within
the mitochondria, remains uncertain.
In recent years, it has become clear that at least two intracellular
molecules assist in mediating the cholesterol transport process. The
first of these, known as the steroidogenic acute regulatory protein
(StAR), is a 30-kDa protein whose synthesis is stimulated by ACTH, and
which associates with mitochondria (1). StAR is believed to act by
promoting the targeted movement of cholesterol to relevant sites on the
outer mitochondrial membrane (1), and is found in all steroidogenic
cells except for those in placenta and brain (1, 2). The second
molecule is actually a complex of molecules collectively known as the
peripheral-type benzodiazepine receptor, or PBR. This membrane-spanning
receptor, found on the outer mitochondrial membrane of all
steroidogenic cells (3), gets its name from its ability to bind natural
and synthetic ligands of the benzodiazepine family. Targeted disruption
of the PBR gene in mouse Leydig tumor cells essentially eliminates
steroidogenic capability (4), which is restored with replacement of an
active PBR complex (4). Unlike StAR, PBR is constitutively expressed in
steroidogenic cells (3). Activation of PBR with endogenous or synthetic
ligands facilitates steroidogenesis, however, suggesting that the
molecule is under regulatory control (3, 5, 6, 7).
If both or either of these regulatory molecules are essential for
steroidogenesis, we hypothesized that the level of expression of StAR
and/or PBR would correlate with changes in steroidogenic capability of
a particular steroidogenic gland during an animal’s life history.
During neonatal life in rats, for example, the adrenal cortex undergoes
a period of about 2 weeks during which constitutive steroidogenesis is
relatively high (compared with cells from adult animals, in which such
activity is absent or negligible) (8, 9), but ACTH-responsiveness is
very low (8, 9, 10, 11). Although changes in ACTH-responsiveness may be partly
related to developmental changes in microsomal steroidogenic enzymes
(12), the mechanisms of both the constitutive process and the lack of
ACTH-responsiveness remains largely unknown. We predicted that StAR
and/or PBR expression would be low during the neonatal period, and that
if true, this could provide an explanation for the reduced
adrenocortical steroid output during this period.
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Materials and Methods
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Animals
Male Sprague Dawley rats were purchased from Holtzman (Madison,
WI). For studies with neonates, timed pregnant females arrived at our
animal care facility at 18 days of gestation. Pups were kept with their
mothers until immediately before use. Both male and female pups were
included in all experiments. All experiments were approved by the
Boston University Institute Animal Care and Use Committee.
ACTH injections
On the day before an experiment, animals were transferred to a
controlled quiet room and given 24 h to acclimate to their
surroundings. All experiments began at approximately 0900–1100 h
(lights on 0700–1900 h). One group of untreated adult male
(
200–250 g) or neonatal (both sexes) rats was killed by
decapitation at the start of the experiment to collect baseline data
for hormone and PBR measurements. Additional groups of rats were
injected ip with 250 µl (adults) or 100 µl (neonates) physiological
saline or porcine ACTH{1–39} (Sigma Chemical Co.) at
a dose of 10 µg/kg, except for postnatal day (pd) 10 pups, which
received 20 µg/kg, and returned to their home cages. These doses were
chosen on the basis of previous experiments (10), and preliminary
trials that indicated their effectiveness in elevating circulating ACTH
to high physiological levels for approximately 2 h. It is unknown
at present why d10 pups required a higher dose of ACTH to achieve the
same circulating levels reached by the lower dose at all other ages
tested, but it is possible that this is related to age-dependent
changes in clearance of ACTH.
Groups of approximately 10 (neonates) or 6 (adults) animals were killed
in random order (saline vs. ACTH-injected) at each of
several selected times after injection. The blood was collected into
EDTA (final concentration 1–2 mM) and centrifuged;
plasma was frozen in aliquots for corticosterone and ACTH RIAs. Adrenal
glands were immediately dissected from the carcasses, decapsulated to
remove the outer glomerulosa/capsule layer, and homogenized on ice for
45 sec in ice-cold Tris (50 mM)/sucrose (0.25
M) buffer, pH 7.4, with a TekMar tissue grinder.
Mitochondria were prepared by differential centrifugation as previously
described (12), and stored frozen for future immunoblot and binding
analyses.
Cell secretion experiments
Adult male (450 g) or neonatal (both sexes; pd 9–11) rats
were killed by decapitation, and the adrenals immediately dissected.
The right adrenal glands from each animal were pooled and used for
in vitro analysis of ACTH sensitivity (see Fig. 1
); the left adrenal glands were pooled,
homogenized as above, and mitochondria prepared for PBR analysis (see
Table 1). For cell secretion studies, the
right adrenals from 12 (adult) or 20–60 (neonates) rats were pooled,
minced, and subjected to enzymatic dispersion as previously described
(9). This process was repeated in three different experiments.
Approximately 100,000–150,000 viable cells with large vacuolar
structures consistent with the appearance of lipid droplets were
incubated in 1 ml of Medium 199 (with 15 µM BSA) (13) for
2 h in a humidified, oxygenated environment. Porcine
ACTH{1–39} or vehicle was then added directly to the tubes in a
volume of 10 µl, and the incubation was continued for 2 additional
hours (9). The cell suspension was then centrifuged at 4 degrees, and
the supernate was collected and frozen for corticosterone RIA.
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Figure 1. Effect of increasing concentrations of ACTH on
corticosterone production from isolated adrenocortical cells. Cells
from adult or pd9–11 adrenal glands were prepared by enzymatic
dispersion as described in Materials and Methods and
preincubated for 2 h. Porcine ACTH{1–39} (or vehicle) at one
of several concentrations was then added to the respective tubes and
the incubation continued for an additional 2 h. The cell
suspension was centrifuged and the supernate assayed for corticosterone
by RIA. Each point is the mean and SEM of observations from
three separate experiments, each performed in duplicate or triplicate.
Results are normalized to the number of viable (dye-excluding), lipid
droplet-containing cells in the suspension. The two curves were
significantly different when assessed by two-factor ANOVA.
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Immunoblot analysis
Adrenal mitochondrial proteins from the unstimulated rats
described above in the injection study, were fractionated by one
dimensional SDS-PAGE on a 15% acrylamide gel. The proteins were then
transferred onto 0.45 µm nitrocellulose membranes (Schleicher &
Schuell, Keene, NH) at 0.9 A for 30 min using a Trans-Blot
Cell (Idea, Corvalis, OR). Nonspecific adsorption of the antibodies was
blocked by incubating the nitrocellulose in 5% milk. The blots were
then treated for immunodetection of PBR, stripped, and reblotted for
detection of StAR protein using anti-PBR and anti-StAR at 1:1000
dilution. Anti-peptide antiserum to amino acids 71–88 of PBR and to
amino acids 89–107 of the 30 kDa StAR protein were prepared as
previously described (14). Goat IgG-horseradish peroxidase was used as
secondary antibody at 1:6000 followed by chemiluminescent detection
(DuPont NEN, Boston, MA). Image analysis of the
immunoreactive protein bands was performed using the Sigmagel software
(Jandel Scientific, San Rafael, CA).
Radioligand binding assays
Additional aliquots of mitochondria (10 µg protein) prepared
above were resuspended in PBS and used for Scatchard analysis of PBR
binding activity. [N-methyl-3H]PK11195
(1-(2-chlorophenyl)-N-methyl-N-(1-methyl-propyl)-3-isoquinolinecarboxamide;
specific activity 83.5 Ci/mmol; DuPont NEN) binding
studies were performed at 4 C, in a final incubation volume of 0.3 ml,
using the radioligand at a concentration range of 0.05–12.5
nM (10–14 concentrations/assay) and 1000-fold excess of
unlabeled ligand (Research Biochemicals International
Inc., Natick, MA), as previously described (5, 14, 15, 16). After 120 min
incubation, assays were stopped by filtration through Brandel GF/C
glass fiber filters and washed with 5 x 5 ml ice-cold PBS.
Radioactivity trapped on the filters was determined by liquid
scintillation spectrometry at 30% counting efficiency. The
dissociation constant (Kd) and the number of binding sites
(Bmax) were determined by Scatchard plot analysis of the
data using the LIGAND program (17) (KELL, version 4.0,
Biosoft, Inc.).
Miscellaneous
Corticosterone and ACTH were determined by RIA according to the
manufacturer’s specifications (ICN), except that volumes of all
reagents were reduced by 50%, and cell suspension supernates were
diluted 1:1000 for adult cells and between 1:10 and 1:100 for neonatal
cells before assay, as previously described (13). Statistical analysis
was by one- or two-factor ANOVA with Bonferonni correction. Regression
analysis and areas under the curve were performed using the Prism
software program from GraphPad, Inc. Protein was determined by the
dye-binding assay of Bradford (18) with 
-globulin as standard.
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Results
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Acutely isolated adrenocortical cells from neonatal (pd9–11) rats
were significantly less sensitive to ACTH in vitro than
cells isolated from adult male rat adrenals (Fig. 1
). In these
experiments, the right adrenals from each animal were pooled within an
experiment for cell isolation and secretion studies, and the left
adrenals were pooled and processed for PBR binding analyses (Table 1).
Mitochondria collected from neonates demonstrated significantly
(P < 0.003) lower PK11195 binding ability and slightly
higher affinity for the ligand (Table 1). Results from the three
experiments (on pd9, 10, and 11, respectively) were pooled for
statistical analyses; interestingly, however, even within this small
window of time there was a trend toward increasing binding ability on
each day (11.5%, 12.6%, and 17.6% of adult binding,
respectively).
To determine if differences in ACTH sensitivity also occurred in
vivo, and to examine other neonatal ages, animals of different
ages were injected once with a dose of ACTH sufficient to elevate
circulating ACTH to high physiological levels. We chose to test animals
on pd5, pd10, and pd15 because at these ages steroidogenesis is
declining, at a nadir, and rebounding, respectively (8, 9). ACTH
injection significantly increased plasma levels of ACTH in all age
groups (Fig. 2). The pattern and maximal
levels of ACTH achieved by the injections were statistically
indistinguishable between ages 5, 10, 15, and adult, except that the
response at pd10 was slightly, but significantly lower than that at
pd15 (P < 0.05), but was not different from other
ages. Despite the similar ACTH profiles following injection, however,
the corticosterone responses to injection were significantly lower in
the neonates (Fig. 2). All neonatal ages demonstrated significantly
(between P < 0.001 and P < 0.05)
lower corticosterone levels after ACTH injection than adults, and the
response on pd10 was significantly lower than at the other two neonatal
ages. Saline injection had no significant effect on plasma levels of
ACTH at any age, but had small, but significant effects on plasma
corticosterone at 30 min on pd15, 120 min on pd5, and 15 min in adults
(Fig. 2).
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Figure 2. Effects of saline or ACTH injection on plasma
levels of corticosterone and ACTH in rats of different ages. Saline or
porcine ACTH{1–39} was injected at time zero as described in
Materials and Methods. A noninjected group of rats was
killed at the start of each experiment for baseline values (zero time
values). "Day" refers to postnatal day. Each point is the mean and
SEM of 6 (adults) and approximately 10 (neonates) animals.
The only significant difference in ACTH-induced ACTH levels, by
2-factor ANOVA, was between pd 10 and 15. ACTH-induced corticosterone
increases were significantly smaller at all neonatal ages when compared
with the response in adults; the response on pd10 was the smallest of
all ages. Saline injection had no effect on ACTH levels, but slightly
and significantly increased corticosterone at 120 min on pd5, 30 min on
pd15, and 15 min in adults.
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In preliminary experiments, it was determined that acute ACTH
injections did not significantly alter PBR expression at any age tested
(not shown). Thus, mitochondria from different timepoints and treatment
groups in the experiments of Fig. 2 were pooled to provide sufficient
protein for use in binding studies and immunoblots. A representative
Scatchard analysis of mitochondrial binding of the synthetic ligand
PK11195 in neonatal and adult rats is shown in Fig. 3 and summarized in Fig. 4. Total binding capacity was higher, and
affinity (Kd) of the mitochondrial receptor for PK11195 was
lower, in mitochondria from adults. When the data from Fig. 4 and Table 1 were pooled, the difference in Kd between pd10 and adult
rats was significant (P < 0.02).
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Figure 3. Representative Scatchard analyses of PBR ligand
binding in mitochondria pooled from pd 9 or adult rats. Mitochondria
were incubated with radiolabeled synthetic ligand as described in
Materials and Methods. Scatchard analysis of multiple
observations was used to quantify maximal binding (Bmax)
and affinity (Kd).
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Figure 4. Summary of PBR ligand binding ability in
adrenocortical mitochondria from rats of different ages (obtained from
the animals in Fig. 2). Values are the mean and SE of at
least three replicates for each age.
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Expression of irPBR, as evidenced by immunoblotting, was 65–80% lower
in neonatal rat adrenals than in adult glands (Fig. 5). Expression of irStAR protein was
highly variable and did not differ significantly between ages (Fig. 5),
although it tended to be highest when PBR expression was lowest. There
was also no effect of acute injections of ACTH on StAR expression at
any age (not shown), although this may have been due to the slight but
significant effects of saline injection on circulating ACTH and
corticosterone in the animals in Fig. 2. Densitometric analysis of
replicate experiments is shown in Fig. 6.
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Figure 5. PBR and StAR expression in adrenal mitochondria
from rats of different ages. Representative pools of mitochondria from
the unstimulated rats in Fig. 2 at each of three neonatal ages or
adults were loaded onto one-dimensional gels and electrophoresed and
blotted as described in Materials and Methods. Bands
corresponding to Mr 18 kDa and 30 kDa were identified as
PBR and StAR, respectively. For comparison, one lane contains
mitochondrial protein isolated from unstimulated MA-10 cells
(progesterone-secreting Leydig cell line). Note that PBR expression is
lowest on pd10. The position of Mr markers (kilodaltons) is
indicated.
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Figure 6. Densitometric analysis of replicate immunoblots
for PBR and StAR proteins. Between 2–4 blots from replicate
experiments, including those in Fig. 5, were analyzed as described in
Materials and Methods and compared with a sample of
mitochondria from adult rat adrenals on each blot (assigned a value of
100%). The results are expressed as mean and SE or range
as percentage of adult value (assigned a value of 100%). *,
P < 0.001 vs. adult.
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The steroidogenic responses to ACTH in Fig. 2 were quantified by
calculating the net area under the curve for corticosterone responses
at each age group. These values were then correlated with levels of
irPBR and PBR Bmax values from Figs. 4 and 6, after first
correcting for nonnormality by transforming the raw data to the arcsin.
The results are shown in Fig. 7. Both
irPBR content and total binding capacity were highly correlated
(r2 = 0.99 for each) with the steroidogenic response to
ACTH in vivo, and irPBR was highly correlated with PBR
binding (Bmax) (r2 = 0.99, not shown).
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Figure 7. Correlation of PBR expression (irPBR), PBR binding
(Bmax), and steroidogenic response to ACTH (area under the
curve from Fig. 2). The correlation coefficients for steroidogenesis
vs. irPBR and Bmax were both 0.99, and were
calculated from the arcsin-transformed data.
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Discussion
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Beginning after the first few postnatal days, the rat adrenal
cortex is relatively unresponsive to stimulation by ACTH and other
secretagogues, reaching a nadir in responsiveness around pd10 (8, 9).
At least part of the hyporesponsiveness of the immature gland may be
related to reduced expression of microsomal enzymes involved in steroid
synthesis (12), although all the steroidogenic enzymes, including
cytochrome P450scc, are expressed in the neonatal rat adrenal at
reasonably high levels when steroidogenesis is at a nadir (12). It is
unlikely, therefore, that reduced expression of steroidogenic enzymes
can account for all or even most of the hyporesponsiveness observed at
this time. Thus, the biochemical basis of ACTH-insensitivity during the
neonatal period in rats (and during fetal life in other species, Ref.
19) remains unclear.
Because previous work suggested that steps distal to generation of cAMP
were the major contributors to stress- and ACTH-hyporesponsiveness of
the neonatal adrenal (9, 12), we examined the ontogeny of regulatory
proteins believed to be essential for cholesterol transport to occur.
The process whereby free cholesterol is transported from the outer to
the inner mitochondrial membrane, where P450scc is located, appears to
involve several steps and at least two regulatory proteins. The first
of these, the steroidogenic acute regulatory protein (StAR), is an
ACTH-inducible, 30 kDa protein expressed in all steroidogenic glands
except the placenta and brain (20). It appears to be required for
delivery of cholesterol to contact sites between the outer and inner
mitochondrial membranes (20). Once there, cholesterol may be
transported through a channel created by activation of a multimeric
protein complex termed the peripheral-type benzodiazepine receptor
(PBR; 3, 20). PBR is an 18-kDa protein localized to the outer membrane,
which in conjunction with an associated voltage-dependent anion
channel, appears to complete the final step of cholesterol delivery to
P450scc (3). Its importance in the steroidogenic process is highlighted
by the observation that targeted disruption of this gene eliminates
steroidogenesis in mouse Leydig tumor cells, an effect that is reversed
upon replacement with functional PBR (4). In addition, pharmacological
inhibition of PBR expression reduces circulating glucocorticoid levels
and decreases adrenocortical cell sensitivity to ACTH (14).
In the present study, expression of irPBR, but not StAR, followed a
developmental pattern that was highly correlated (r2 =
0.99) with the developmental pattern of steroidogenesis. Immediately
following birth, stress- and ACTH-induced adrenocortical
steroidogenesis is relatively high (8, 9, 10, 11, 12, 21, 22), and then steadily
declines over a period of approximately 5–6 days, reaching a nadir
around pd10 (8, 9, 21, 22). By approximately pd15, ACTH-sensitivity
begins to return. irPBR and PBR ligand binding capacity followed this
pattern of development, and were highly correlated with
steroidogenesis. StAR protein was constitutively expressed at
comparable levels in neonatal and adult adrenals, and did not appear to
change 2 h following ACTH injection. It is possible that the mild
stressor of saline injection was sufficient to elevate resting StAR
levels so as to obscure a response to ACTH. In any event, it appears
from the present results that expression of PBR, not StAR, is the
primary limiting factor in the ontogenic increase in steroidogenesis in
rat adrenal.
The affinity of PBR for the synthetic ligand PK11195 was slightly
higher in neonatal adrenal mitochondria than in adults, perhaps
suggesting that the receptor is posttranslationally modified in
neonates. Although we do not yet know the nature of this putative
modification, the higher affinity of the neonatal receptor could
conceivably contribute to the relatively high constitutive
steroidogenesis that is characteristic of neonatal rat adrenal cells
(8, 9). For example, if the receptor were activated by low (basal)
levels of endogenous PBR ligands, such as the endozapine
diazepam binding inhibitor (3, 20), this could facilitate
steroidogenesis even in the absence of ACTH. By contrast, because the
total number of binding sites is reduced, maximal steroidogenesis would
be expected to be only a fraction of that found in adult glands. The
physiological significance of constitutive steroidogenesis, which is
unique to fetal and neonatal adrenals and is largely lost upon
differentiation (8, 9), may be related to the requirement for low, but
constant circulating levels of glucocorticoids during fetal/neonatal
development (23). High (i.e. ACTH-induced) levels of
glucocorticoids are neurotoxic during development, but constitutive
levels appear to be required for proper neural and systemic
differentiation (23, 24, 25). Thus, PBR may be a key regulator within the
adrenal gland that allows proper titration of circulating
glucocorticoids during critical windows of development. In addition, it
is also possible that the relatively high expression of StAR protein on
pd10 contributes to constitutive steroidogenesis.
The reduced sensitivity of neonatal rat adrenals to ACTH was maintained
in vitro after dispersion of adrenocortical cells,
suggesting that cellular insensitivity to ACTH in vivo did
not result from putative circulating inhibitory factors that interfered
with ACTH action. In those experiments, adrenals used for PBR binding
assays revealed a trend toward increasing activity (Bmax)
across a limited age span from pd9–11, suggesting that this is a key
window in the developmental expression of PBR. Whether PBR expression
is developmentally suppressed before this time, or is induced
thereafter, remains uncertain. We have previously demonstrated that
chronic exposure of neonatal rats to ACTH during the hyporesponsive
period results in accelerated maturation of the steroidogenic response
to ACTH (10). Similar results have been demonstrated in fetal sheep
(19). Based on the correlation between PBR and ACTH-inducible
steroidogenesis in the present study, we predict that such treatment
would induce a coincident increase in PBR expression and activity as
well.
It is likely that proteins other than PBR follow a developmental
pattern similar to the one identified in this study. For example, one
of us has recently determined that cytochrome P4501B1, a
xenobiotic-metabolizing adrenal enzyme, follows a very similar profile
to that of PBR during neonatal ages in rats (26). Nonetheless, PBR is
the only regulatory factor known to be important for ACTH-induced
steroid synthesis that displays a developmental pattern similar to that
of steroidogenesis. Thus, we believe that it is the expression of this
important cholesterol-transporter that is the primary factor limiting
steroidogenesis in the neonatal period in rats. Furthermore, the
neonatal rat may provide a valuable model for determining those factors
that regulate PBR expression in vivo. It should be
emphasized, however, that neonatal ACTH-insensitivity is likely to be a
complex phenomenon involving not only changes in cholesterol transport
capacity, but changes in cytochrome P450 expression or activity, and
possibly in cAMP-independent signalling pathways such as
calcium/calmodulin and protein kinase C.
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Acknowledgments
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The authors gratefully acknowledge the assistance of Ms. Andrea
Corrente.
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Footnotes
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1 This work was supported in part by NSF Grant IBN9513926 (to E.P.W.),
and NIH Grants RO1-ES07747 (to V.P.) and K04-HD01031 (to V.P.). 
Received May 7, 1998.
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Abstract
Introduction
