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Endocrine (H.R., B.M.J., E.D.B.) and Immunology (M.K.O.) Research Laboratories, St. Luke’s Medical Center, Medical College of Wisconsin, Milwaukee, Wisconsin 53215; and the Department of Surgery, University of Minnesota Medical School (W.C.E.), Minneapolis, Minnesota 55455
Address all correspondence and requests for reprints to: Hershel Raff, Ph.D., St. Luke’s Health Science Building, 2901 West KK River Parkway, Suite 503, Milwaukee, Wisconsin 53215. E-mail: hraff{at}mcw.edu
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Abstract |
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Neonatal rats (with their lactating dams) were exposed to normoxia (21% O2) or hypoxia (12% O2) continuously for 7 days from birth. Trunk blood was collected, and entire adrenal glands were processed from 7-day-old rats to study the activity of the steroidogenic pathway in dispersed cells and isolated mitochondria, for measurement of expression of the steroidogenic enzyme messenger RNAs (mRNAs) by RT-competitive PCR and in situ hybridization histochemistry, for measurement of zona glomerulosa width by immunohistofluorescent staining for P450c11AS protein, and for measurement of mitochondrial number and distribution by transmission electron microscopy.
Exposure to hypoxia for 7 days from birth resulted in a marked increase in plasma ACTH, corticosterone, and aldosterone with no change in PRA. Aldosteronogenesis and P450c11AS activity were both augmented in dispersed cells; this effect was lost in isolated mitochondria (from entire adrenal glands) using a permeable substrate for P450c11AS. There was no significant effect of hypoxia on expression of the steroidogenic enzyme mRNAs measured by RT-competitive PCR or in situ hybridization histochemistry. Finally, hypoxia had no effect on mitochondrial number or stereology as assessed by transmission electron microscopy or on zona glomerulosa width as assessed by staining for P450c11AS protein.
We conclude that, as opposed to that in adults, hypoxia in the neonate results in an augmentation of aldosteronogenesis. This effect is not accounted for by a change in steroidogenic enzyme mRNA expression, zona glomerulosa width (i.e. hyperplasia), or mitochondrial number or distribution. This functional augmentation of aldosteronogenesis may be due to a change in mitochondrial permeability to steroid substrates and/or the effect of cytosolic factors that control mitochondrial steroidogenesis.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Whereas the ontogeny of the glucocorticoid pathway has been carefully examined in the rat (20, 21), only recently did Feuillan and Aguilera carefully examine the regulation of the aldosterone pathway and enzyme expression in the 7-day-old rat (22). Whereas the regulation of the glucocorticoid pathway was similar to that in the adult, administration of dexamethasone (which suppressed endogenous ACTH release) resulted in a marked decrease in aldosteronogenesis in vitro that was only partially restored by ACTH administration. Administration of ACTH per se resulted in a decrease in aldosterone synthase (P450c11AS) activity, as has been reported in the adult (23, 24). This apparent asymmetry suggests that normal basal ACTH release is necessary for the maintenance of aldosteronogenesis in the 7-day-old rat, but that ACTH stimulation of the zona glomerulosa (ZG) can actually decrease P450c11AS expression.
Thomas and Marshall have developed a model for the exposure of rats to hypoxia from birth by exposing pregnant rats to hypoxia from the last 2 days of gestation through parturition to as much as 54 days of age (25). In the present study, we have modified their method such that neonatal rats with their lactating dams are exposed to hypoxia just after parturition continuously until they are studied at 7 days of age.
By combining the approach of Feuillan and Aguilera in the 7-day-old rat adrenal with the methods of exposure to hypoxia of Thomas and Marshall, the present study addresses the following questions. What are the effects of exposure to hypoxia from birth to 7 days of age on 1) plasma ACTH, renin activity, aldosterone, and corticosterone; 2) steroidogenesis in vitro; 3) expression of steroidogenic enzyme messenger RNAs (mRNAs); 4) ZG width; and 5) mitochondrial density of the cells of the ZG? Therefore, this study presents a characterization of the physiological, biochemical, microanatomical, ultrastructural, and molecular adaptation of the adrenal cortex to hypoxia in the neonatal rat.
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Materials and Methods |
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Lactating dams were maintained with their litters for 7 days in a normoxic or hypoxic environment (25). Chambers were opened on day 4 to clean the cages. At 0800 h on day 7, dams were quickly removed from the chambers. Then, rat pups were quickly decapitated, and trunk blood from three or four pups was pooled for the measurement of plasma ACTH, renin activity, aldosterone, and corticosterone. Adrenal glands were quickly removed and randomly assigned for processing for each technique described below. Adrenal glands for PCR and in situ hybridization histochemistry (ISHH) were always frozen first.
Six experimental days (1 normoxic vs. 1 hypoxic litter/day; 12 litters total) were required to collect sufficient plasma volume for hormone measurements and for the number of adrenal glands necessary for the following: 4 different dispersed cell preparations on 4 different days, RT-PCR and ISHH of steroidogenic enzymes, assessment of ZG width by staining for P450c11AS protein, and electron microscopy. Another 10 experimental days (1 normoxic litter vs. 1 hypoxic litter; 20 litters total) were required for analysis of enzyme activity in isolated adrenocortical mitochondria (10 different mitochondrial preparations on 10 different days).
Dispersed cells
Whole adrenal glands were minced and dispersed with collagenase
(Worthington Biochemical Corp., Freehold, NJ). Although
Feuillan and Aguilera (22) were able to separate the capsule (ZG) from
the subcapsule [zona fasciculata (ZF)/zona reticularis
(ZR)] in 7-day-old rat adrenals, we were unable to get a sufficiently
clean separation of adrenals from hypoxic 7-day-old rats to generate
sufficient capsular (ZG) cells to perform the experiments described.
Therefore, P450scc activity represents the early pathway from all
adrenal zones. The dispersed cells were washed and placed in cold
Krebs-HEPES-calcium buffer at a concentration of 50,000 cells/ml. Cells
were always studied the day they were dispersed. Cells were placed in
test tubes and incubated for 2 h at 37 C in a shaking water bath,
and activities of the different mitochondrial enzymatic steps of the
steroidogenic pathways were assessed as described previously (28). Each
treatment within an experimental day (basal, cAMP, cyanoketone) was
performed in triplicate. Briefly, the entire aldosterone (ZG) and
corticosterone (ZF/ZR) pathways were assessed by stimulation with
(Bu)2cAMP (3 mM), P450scc activity (all zones)
was assessed by stimulation of the conversion of endogenous cholesterol
to pregnenolone with cAMP in the presence of cyanoketone
(3ß-hydroxysteroid dehydrogenase inhibitor; 10 µM)
generously donated by Sterling-Winthrop (Collegeville, PA), and
P450c11AS activity (ZG) was measured by the conversion of
corticosterone (7.2 µM; Sigma Chemical Co.,
St. Louis, MO) to aldosterone in the presence of cyanoketone.
Triplicate replications for each treatment on an experimental day were
averaged and treated as one value. The n values in the figure legends
represent the numbers of different cell preparations (experimental
days).
Isolated mitochondria
Mitochondria were isolated by a modification of our previously
published method (29). Whole adrenal glands were minced on ice and
washed in ice-cold Tris-sucrose buffer. Tissue was homogenized in
ice-cold Tris-sucrose buffer using a Potter-Elvehjem tissue grinder on
ice. Homogenized tissue was centrifuged at 900 x g for
10 min at 4 C, and the supernatant was transferred to a new ice-cold
tube and centrifuged at 9000 x g for 10 min at 4 C.
The supernatant was discarded, and the pellet was resuspended in
ice-cold buffer (Tris-HCl, sucrose, KCl,
K2HPO4, MgCl2, and 0.2% BSA,
pH 7.5) to a concentration of 1 mg mitochondrial protein/ml. P450scc
activity (all zones) was assessed by the addition of 10 mM
isocitrate, 1 µM cyanoketone, and 1 ng/ml
25-hydroxycholesterol (Sigma Chemical Co.), incubated at
37 C, with the mixture sampled at 0 and 30 min for the measurement of
pregnenolone. P450c11AS activity (ZG) was measured by the addition of
10 mM isocitrate and 7.2 µM corticosterone
for the measurement of aldosterone at 0 and 30 min. The two steroid
substrates were dissolved in ethanol such that the final concentration
of ethanol was 0.1%. The n values in the figure legends are the
numbers of different mitochondrial preparations (e.g.
experimental days).
RT-competitive PCR (RT-cPCR) of P450scc, P450c11B, and P450c11AS
mRNA
Whole adrenal glands were quickly frozen and stored in liquid
nitrogen. The RNA extraction, RT, PCR primers, and PCR competitive
mimics have all been described in detail previously (28, 30). Briefly,
total cellular RNA was extracted with guanidine thiocyanate and single
strand complementary DNA generated using Superscipt preamplification
reagents (Life Technologies, Gaithersburg, MD). cPCR was
performed with specific primers for P450c11AS (CYP11B2), P450c11B
(CYP11B1), and P450scc (CYP11A1) using published sequences (31, 32, 33) and
specific mimics we constructed previously (28) using a commercial kit
(CLONTECH Laboratories, Inc., Palo Alto, CA). PCR products
were separated by electrophoresis in 2% agarose gel and analyzed by
ethidium bromide staining and a CCD camera/video gel
documentation/image analysis system and software (Bio-Rad Laboratories, Inc., Hercules, CA). The quantity of each target
mRNA was estimated by assessing the quantity of mimic that produced
equimolar amplification (28).
ISHH of steroidogenic enzyme mRNA
Adrenal glands were quickly frozen in isopentane tubes in liquid
nitrogen. The ISHH procedure we used has been previously described in
detail (28, 34, 35). Briefly, adrenal glands were frozen-sectioned and
mounted, and adjacent sections were hybridized using oligonucleotide
probes. Measurements of hybridization area and density were analyzed by
UMAX scanner, with hybridization area and density used as indexes of
mRNA levels in adjacent tissue sections. The resolution of the
autoradiographs was not sufficient to distinguish P450scc in the ZG
vs. the ZF.
Ultrastructural analysis
Mitochondrial density of ZG cells was estimated by counting
sections analyzed by transmission electron microscopy. Cells were
identified by their characteristic location and morphometry (36).
Approximately 40 sections/adrenal gland were randomly counted
regardless of the number of cells represented. The results, therefore,
are presented as the number of mitochondria per 550 µm2.
Otherwise, mitochondria were counted as described previously (37).
ZG width
Immunohistofluorescent staining for P450c11AS protein was
performed on adrenal sections as described previously (38). Briefly,
adrenal sections were fixed with Zamboni’s fixative, blocked with
normal donkey serum, and incubated overnight with rabbit anti-P450c11AS
antibody (provided by C. Gomez-Sanchez, University of Missouri,
Columbia, MO). Sections were incubated with Cy3-labeled donkey
antirabbit secondary antibody and coverslipped. Control slides
incubated with the anti-P450c11AS antibody preabsorbed with a truncated
version of the immunizing peptide were negative for staining. Optical
images were collected using a CCD camera. Using the presence of
P450c11AS protein staining to define the zona glomerulosa, the size of
the zone was estimated for each adrenal (4–6 adrenals from normoxic
and hypoxic pups) by measuring the average width of a minimum of 10
sections taken from the central region of each adrenal.
Hormone/protein assays
Plasma ACTH, renin activity, corticosterone, and aldosterone
were measured by RIA (26, 27). The concentrations of pregnenolone,
corticosterone, and aldosterone in cell dispersion and isolated
mitochondria experiments were measured by RIA (28, 29). Mitochondrial
protein concentration was measured by UV spectrometry (Milton Ray,
Rochester, NY).
Statistical analysis
Data were analyzed by unpaired t test, two-way ANOVA,
Duncan’s multiple range test, and Mann-Whitney test. Replicates on
each experimental day were averaged and treated as one datum; n values
in each figure legend are the numbers of different cell or
mitochondrial preparations. P < 0.05 was considered
significant. Data are presented as the mean ±
SEM.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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shows the plasma hormone
levels measured at the end of 7 days of exposure to normoxic or hypoxia
in 7-day-old rat pups (three or four pups per sample). Exposure to
hypoxia for 7 days resulted in significant increases in plasma ACTH,
corticosterone, and aldosterone without a significant change in
PRA.
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shows the results of
steroidogenesis measured in dispersed cells from 7-day-old rat pups
exposed to normoxia or hypoxia for 7 days. Cells from hypoxic rats
exhibited increased basal and cAMP-stimulated aldosterone production
in vitro. However, corticosterone production in
vitro was not altered by exposure to hypoxia.
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). We were not able to adequately
separate the capsule and subcapsule from 7-day-old hypoxic pups to
generate sufficient capsular cells for a separate analysis of P450scc
activity in ZG vs. ZF/ZR cells. Therefore, P450scc activity
is from the entire adrenal cortex. Cells from hypoxic rat pups
exhibited increased pregnenolone and aldosterone production in the
presence of cyanoketone.
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).
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We then assessed whether exposure to hypoxia might alter the expression
of the genes for the steroidogenic enzymes, as determined by
semiquantitative assays for steady state mRNA levels. Figure 5 shows representative digitized gels of
RT-cPCR for the three mitochondrial enzymes. mRNA concentrations were
calculated by determining the concentration of added PCR mimic
(lower band) that yields a target to mimic ratio of 1.
Figure 6 shows the combined results of
these experiments. Although there was a tendency for P450scc mRNA (all
zones) to be higher in cells from hypoxic rats, there were no
statistically significant differences. There were no detectable changes
in P450c11B or P450c11AS mRNAs as determined by this sensitive assay.
To confirm these findings using an alternate approach and to assure
that hypoxic did not induce ectopic (ZF) expression of P450c11AS, we
assessed mRNAs of all five steroidogenic enzymes by ISHH.
Representative autoradiographs, shown in Fig. 7, illustrate no obvious differences in
the intensity or location of expression of the three regulated
mitochondrial enzyme mRNAs. We were unable to resolve P450scc mRNA in
ZG vs. ZR in individual autoradiographs. Quantitative
assessment of the combined ISHH data are shown in Fig. 8. There were no significant
differences between adrenals from normoxic vs. hypoxic rats
for any of the mitochondrial or microsomal steroidogenic enzymes.
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shows representative images of
immunohistofluorescent staining for P450c11AS protein to assess the
width of the ZG in adrenals from normoxic and hypoxic pups. There were
no apparent effects of hypoxia, nor was there a statistical difference
when the composite of images for many sections was evaluated. ZG width
averaged 49.0 ± 1.5 µm in four adrenals from normoxic rats
vs. 45.7 ± 1.8 µm in six adrenals from hypoxic
rats.
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Discussion |
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The increase in plasma ACTH in the neonate was much more profound than we have demonstrated previously for adult rats, even those exposed to more severe decreases in inspired oxygen (26). It was also surprising that such a large increase in ACTH was generated considering the well described stress hyporesponsive period of the neonatal/infant rat pup (39, 40). This unique nature of neonatal hypoxia, not previously studied to our knowledge, is an exciting finding that we are currently pursuing.
It is not surprising that this large increase in ACTH generated an increase in plasma corticosterone (20, 21), albeit a small one compared with that in adult animals. However, that the presumably chronic increase in ACTH also resulted in marked increases in aldosterone is surprising because exogenous ACTH has been shown to decrease the expression of P450c11AS in adults and 7-day-old rats (22, 23, 24). There are several possible explanations for this. The first is that plasma ACTH measured at the time of decapitation was an acute response to handling and decapitation and was not representative of ACTH levels during the previous few days. The effect of handling is probably not responsible per se, because normoxic neonates handled in the same way had low basal ACTH and corticosterone levels. Another possibility is that hypoxia-generated increases in ACTH and, perhaps, some other factor, maintain aldosteronogenesis in the neonate and are a more realistic representation of stress-induced adrenal steroidogenesis than periodic ACTH injections. In the adult rat, chronic stress (immobilization or repeated NaCl injection ip) has been shown to decrease aldosteronogensis and P450c11AS expression (41). This suggests that the neonate has developed a unique adaptation to hypoxic stress to maintain aldosterone production when confronted by an increase in ACTH. The lack of an increase in PRA in the 7-day-old hypoxic rat suggests that increased plasma aldosterone levels were not due to the action of angiotensin II. This is supported by the finding in the human neonate that increases in aldosterone are not dependent on PRA and that ACTH plays a dominant role (42, 43).
What might be the mechanism of increased aldosterone production characterized by an increase in P450c11AS (late pathway) activity? It would have been desirable to separate capsules and subcapsules to examine P450scc activity in ZG vs. ZF/ZR. As stated previously, we were unable to obtain a clean separation of capsules and subcapsules in adrenals from hypoxic 7-day-old rats sufficient to generate enough cells quickly enough to perform this analysis. It is likely, on the basis of zone size alone, that most of the P450scc activity or mRNA that we measured in whole cells, mitochondria, and PCR represents ZF/ZR. Despite this, the main finding of increased aldosteronogenesis and P450c11AS activity (from ZG cells) with no change in corticosterone production (from ZF) or P450c11AS mRNA (ZG) remains valid and compelling.
At first, we hypothesized that a cytosolic factor might increase the rate-limiting step of steroidogenesis: cholesterol translocation from the outer to the inner membrane of the mitochondria. Two cytosolic proteins have been proposed that might be appropriate: steroidogenic acute regulatory protein and the peripheral benzodiazepine receptor (44, 45). As there appears to be no evidence that steroidogenic acute regulatory protein or peripheral benzodiazepine receptor augments the entry of corticosterone or deoxycorticosterone into the mitochondria, our experiments with cyanoketone (3ß-hydroxysteroid dehydrogenase inhibitor) and exogenous corticosterone to aldosterone conversion argue against this possibility. On the other hand, the production of pregnenolone from exogenous 25-hydroxycholesterol, which is freely permeable through the mitochondrial membrane, was not augmented, suggesting that this may be a cholesterol transport/mitochondrial translocation mechanism. It may also be that hypoxia increases the production of a cytosolic factor and/or a change in cytosolic milieu (e.g. calcium or pH) that augments passage of steroids through the mitochondrial membrane (46, 47, 48, 49). It may also be that the rate of steroidogenesis (e.g. Vmax) was altered by hypoxia and that the increase in aldosteronogenesis in whole cells might have been demonstrated in mitochondria had a longer incubation time been used.
That basal aldosteronogenesis was increased is worthy of consideration in more detail. What if exposure to hypoxia in vivo, by increasing ACTH input to the adrenal gland, resulted in an increase in substrate available for conversion after the cells were dispersed? A longer preincubation time may have prevented this. Arguing against this effect is that addition of excess exogenous substrate for P450c11AS still resulted in augmented aldosterone production. Also arguing against this is the finding that basal corticosterone production, which is highly ACTH dependent, was not augmented. Yet another area to be considered are changes in bioenergetics within the adrenal cell. Perhaps hypoxia selectively altered the redox state (NADPH/NADP) and/or cAMP generation within the ZG (but not ZF) cell, leading to a more effective use of available substrate (50).
Another possibility is that exposure to hypoxia from birth to 7 days resulted in an increase in glomerulosa cell mitochondrial density/number or zonal width (i.e. hyperplasia). The lack of an effect of hypoxia on mitochondrial protein content and morphometric analysis of ZG cell mitochondrial number and distribution weighed heavily against this possibility. There was also no statistical difference in the width of the ZG, as assessed by immunohistofluorescent staining for P450c11AS protein.
Finally, we expected that changes in steroidogenic enzyme activity would reflect changes in steroidogenic enzyme expression as reflected by mRNA levels. We could find no evidence for such an effect using two independent methods (RT-PCR and ISHH) with independent sets of probes. This is quite different from our previous studies in adult rats, in which aldosteronogenesis, P450c11AS activity, and P450c11AS mRNA levels all decreased after exposure to hypoxia (28). We can only surmise from this that control of aldosteronogenesis and steroidogenic enzyme expression during hypoxia is quite different in the neonate compared with that in the adult.
This work has both basic and clinical significance. The possibility of a hypoxia-induced cytosolic factor that facilitates steroid flux across the mitochondrial membrane is exciting and certainly bears further investigation. As increasing steroidogenesis is vital for the neonate to survive a hypoxic episode (11, 12, 13, 14, 15, 16, 17, 18, 19), any form of adrenal insufficiency, even if relatively mild, would certainly render the hypoxic neonate susceptible to significant morbidity and mortality.
In the hypoxic adult, a decrease in aldosterone secretion normally occurs and is advantageous by preventing the development of pulmonary and cerebral edema by allowing diuresis and natriuresis (51, 52). However, the control of blood volume is considerably more tenuous in the newborn (3). An increase in aldosterone is probably advantageous in the hypoxic newborn by promoting sodium and water retention and thereby preventing hypovolemia. The importance of increased aldosterone in the maintenance of sodium balance and blood volume has clearly been demonstrated in normal newborns and infants (53, 54), newborns with asphyxia (55), premature newborns (56, 57), and infants with congestive heart failure or respiratory distress syndrome (58, 59).
In summary, whereas an increase in aldosterone is detrimental to the adaptation to hypoxia in the adult, it probably prevents the loss of sodium, hypovolemia, and hyperkalemia in the newborn. Our data suggest that the newborn ZG exhibits a unique adaptation to hypoxia, which, compared with other stressors studied and despite an increase in plasma ACTH, allows an increase in aldosteronogenesis and a prevention of P450c11AS down-regulation. The existence and identification of the intracellular factors/mechanisms responsible for the augmentation of aldosterone synthesis during hypoxia in the neonate may lead to new therapies for the treatment of adrenal dysfunction and critical illness in the newborn.
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Footnotes |
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Received November 3, 1998.
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