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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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Introduction |
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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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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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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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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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). 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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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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). 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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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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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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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Footnotes |
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Received May 7, 1998.
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