Endocrinology Vol. 138, No. 6 2354-2358
Copyright © 1997 by The Endocrine Society
Effects of Gestation on Enzymes Controlling Aldosterone Synthesis in the Rat Adrenal1
Michèle Brochu2,
Jean-Guy Lehoux and
Serge Picard
Research Center, Sainte-Justine’s Hospital (M.B., S.P.), and the
Department of Obstetrics-Gynecology, University of Montreal, Montreal,
Quebec, Canada H3T 1C5; and the Department of Biochemistry, Faculty of
Medicine, University of Sherbrooke (J.G.L.), Sherbrooke, Quebec, Canada
J1H 5N4
Address all correspondence and requests for reprints to: Michèle Brochu, Ph.D., Research Center, Ste-Justine’s Hospital, 3175 Côte Ste-Catherine, Montreal, Quebec, Canada H3T 1C5. address: brochum{at}ere.umontreal.ca
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Abstract
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In the present study, the effects of gestation on various enzymes
implicated in corticosteroid synthesis were evaluated in adrenal zona
glomerulosa and zona fasciculata-reticularis of the Sprague-Dawley rat.
The activity and expression of cholesterol side-chain cleavage
cytochrome P450, 11ß-hydroxylase cytochrome P450, and aldosterone
synthase cytochrome P450 (P450aldo) were analyzed. Plasma aldosterone
levels were increased significantly at 22 days gestation (n = 10)
and fell below the nonpregnant levels at 18–36 h postpartum (n =
11). The activity and expression of 11ß-hydroxylase cytochrome P450
and cholesterol side-chain cleavage cytochrome P450 were not modified
by gestation. P450aldo activity increased at 14 days gestation (n
= 4) and returned to the prepregnancy level at 2 weeks postpartum
(n = 5). As shown by Northern blot analysis (n = 3), P450aldo
messenger RNA increased significantly at 22 days gestation and
decreased 18–36 h postpartum. We clearly demonstrated that elevated
plasma aldosterone levels during pregnancy are associated with
augmented activity and messenger RNA levels of P450aldo in the zona
glomerulosa.
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Introduction
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ALDOSTERONE is the most potent
steroid-regulating electrolyte balance. It is well known that pregnancy
is associated with a substantial increase in circulating aldosterone in
rats (1, 2) and humans (3, 4).
There are two main regulatory sites of aldosterone biosynthesis, the
early rate-limiting step, which is the conversion of cholesterol to
pregnenolone, and the final steps, which involve the transformation of
deoxycorticosterone to aldosterone. The mitochondrial cholesterol
side-chain cleavage cytochrome P450scc (P450scc) is responsible for
pregnenolone formation. Two specific cytochromes P450 are involved in
the final steps of corticosteroid biosynthesis: aldosterone synthase
(P450aldo) and 11ß-hydroxylase (P45011ß). These two enzymes have
been isolated and purified (5, 6). They are present in the
mitochondrial fraction of the adrenal zona glomerulosa for P450aldo and
mainly in the zona fasciculata-reticularis for P45011ß (7, 8, 9).
However, some researchers have found P45011ß messenger RNA (mRNA) in
the entire adrenocortical zone (10, 11). Complementary DNAs (cDNAs) for
these two cytochromes P450 have been cloned (12, 13). Four forms of rat
P45011ß genes have been isolated and characterized (14). CYP11B1 and
CYP11B2 genes encode P45011ß and P450aldo, respectively. CYP11B4
appears to be a pseudogene, whereas CYP11B3 resembles CYP11B1. Mellon
et al. (15) reported that CYP11B3 is expressed only in the
newborn adrenal gland and not in the fetal or adult gland. However,
Zhou et al. (16) demonstrated reverse transcription-PCR
detection of CYP11B3 mRNA expressed in adult rat adrenal.
We have already shown that aldosterone secretion is increased in
adrenal cortex preparations derived from 22-day pregnant rats (17). The
mechanisms by which aldosterone secretion is regulated during
normotensive pregnancy have yet to be clarified. In the present report,
we investigated the effects of pregnancy on the enzymes controlling
aldosterone synthesis: P450scc, P450aldo, and P45011ß.
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Materials and Methods
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Animals
Female Sprague-Dawley rats (Charles River Canada, St. Constant,
Canada), weighing 225–250 g, were mated with males. The morning on
which spermatozoa were found in vaginal smears was deemed to be day 1
of pregnancy. The experiments were performed on days 7, 14, and 22
(term) of gestation and at 18–36 h and 2 weeks postpartum. In the
postpartum period, the rats were nursing their pups. Nonpregnant rats
picked randomly during the estrous cycle served as controls. All
animals were housed under controlled light (lights on from 0600–1800
h) and temperature (21 ± 3 C). They were fed a normal synthetic
diet (Rodent chow, Charles River Canada), containing 190 mEq/kg sodium
and 210 mEq/kg potassium, and tap water ad libitum. This
study received approval from the local animal care committee, which is
accredited by the Canadian Council on Animal Care. Animals were killed
by decapitation (between 0900–0930 h), and trunk blood was rapidly
collected for plasma steroid measurements. The adrenals were picked up
for enzyme assays or enzyme expression analysis.
Enzyme assays in adrenal cortex preparations
Adrenals were harvested immediately after decapitation. Capsules
containing the zona glomerulosa were separated from the zona
fasciculata-reticularis attached to medulla by manual compression.
Adrenal cortex preparations were made as described previously (17). In
brief, both capsules from each rat were equilibrated for 120 min in 5
ml Ham’s F-12 medium (Life Technologies, Burlington, Canada) with
0.2% BSA (Sigma Chemical Co., Mississauga, Canada) and 1.25
mM Ca2+ at 37 C in 5% CO2. After
equilibration, the capsules were transferred to 2-ml wells containing 1
ml Ham’s F-12 medium and the substrate. The same protocol was used for
the zona fasciculata-reticularis (containing the medulla). For enzyme
assays, the substrate concentrations used were saturating, and the
rates of product formation were linear with respect to incubation times
in the adrenal cortex preparations or the zona fasciculata-reticularis
preparations. The substrates employed were 20 µM
corticosterone for P450aldo activity and 40 µM
deoxycorticosterone for the P45011ß activity. Samples (50 µl) were
collected at 0, 2, 4, 6, 8, and 10 min and replaced with fresh medium.
For the determination of P450scc activity, transformation of endogenous
cholesterol to pregnenolone was measured in the presence of 1
µM trilostane (Sterling Wintrop, Rensselaer, NY), and
samples (50 µl) were collected at 0, 5, 10, and 15 min. Trislostane,
an inhibitor of 3ß-hydroxysteroid dehydrogenase, was added to avoid
the rapid conversion of pregnenolone to progesterone. The steroids
formed (aldosterone for P450aldo activity, corticosterone for P45011ß
activity, and pregnenolone for P450scc activity) were measured directly
by RIA as described previously (18) or with RIA kit from ICN
Biochemicals (Immunocorp, Montreal, Canada). The antibodies used were
highly specific. At 50% displacement, the antiserum for aldosterone
cross-reacts 0.09% with deoxycorticosterone, 0.04% with
corticosterone, and less than 0.01% with the other steroids. The
antiserum for pregnenolone cross-reacts 100% with pregnenolone
sulfate, 3.1% with progesterone, 0.85% with 5
-dihydroprogesterone,
and less than 0.02% with the other steroids. Finally, the antibody
against corticosterone has 0.34% cross-reaction with
deoxycorticosterone, 0.10% with testosterone, 0.05% with cortisol,
and less than 0.02% with the other steroids.
RNA analyses
Total RNA was extracted from the adrenal zona glomerulosa and
zona fasciculata-reticularis (containing the medulla) of pregnant and
nonpregnant rats by a modification of the method of Chomczynski and
Sacchi (19) using TRIzol reagent (Life Technologies). Final RNA pellets
were dissolved in diethyl pyrocarbonate-treated water and stored at
-20 C. All RNA concentrations were determined by measuring absorbance
at a wavelength of 260 nm. Each RNA extract was made from the adrenals
from two or three rats from each group and was used for one
experiment.
Total RNA samples were denatured by heating at 60 C in buffer
containing 50% deionized formamide, 10 mM
4-morpholinepropanesulfonic acid (MOPS), and 17% formaldehyde. RNAs
(25 µg total RNA) were separated by electrophoresis in 1.2%
agarose-2.5% formaldehyde gel submerged in buffer (pH 7.0) containing
10 mM MOPS, 40 mM sodium acetate, and 5
mM EDTA. Separate RNAs were transferred to nylon membranes
(Hybond-N, Amersham Canada, Oakville, Canada) using the standard
capillary technique with 10 x SSC (1 x SSC = 0.15
M NaCl and 0.015 M sodium citrate, pH 7) and
fixed under a UV lamp (Gene Linker, Bio-Rad, Mississauga, Canada).
Prehybridizations were carried out at 42 C in buffer composed of 25
mM KPO4 (pH 7, 4), 1 mg/ml Ficoll, 1 mg/ml BSA,
1 mg/ml polyvinyl pyrrolidone, 1% SDS, 50% deionized formamide,
5 x SSC, and 250 µg/ml denatured herring sperm DNA (Boehringer
Mannheim, Laval, Canada). The blots were hybridized with P450scc cDNA,
obtained from Dr. Y. Tremblay (Laboratory of Molecular Endocrinology,
CHUL, Ste-Foy, Canada) and with oligonucleotide probes specific for
each P450 mRNA: a 20-mer for P45011ß and a 35-mer for P450aldo (20).
Specific insert coding for human P450scc (21) was obtained by digestion
of the plasmid pUC18 with EcoRI and purification by
electrophoresis. This cDNA was labeled with
[32P]deoxy-CTP using the Multiprime labeling kit from
Amersham Canada. Oligonucleotides were prepared by the Sheldon
Biotechnology Center (McGill University, Montreal, Canada). They were
end labeled with [
-32P]ATP using polynucleotide kinase
(BRL, Burlington, Canada). Hybridizations were then performed at 42 C
for 24 h in prehybridization buffer to which were added the
labeled probe and 10% (wt/vol) dextran sulfate. To reduce further
cross-hybridization between the oligonucleotide and P450aldo or
P45011ß, an excess of unlabeled oligonucleotide corresponding to the
other mRNA was added to the hybridization solution. The membranes were
then washed in 2 x SSC at room temperature and in 2 x
SSC-1% SDS (wt/vol) at 60 C. Washed membranes were exposed to
autoradiography film (Reflection, DuPont, Montreal, Canada) with
intensifying screens at -80 C for 3–10 days. Hybridization signals
were quantified with the use of a laser densitometer (LKB
Ultrascan XL, Pharmacia, Dorval, Canada). Steady state mRNA levels
were expressed as arbitrary densitometric units and standardized by
comparison with hybridization results obtained with random
prime-labeled 18S ribosomal RNA. Sequential hybridizations were
performed on the same membrane with each probe.
Plasma aldosterone and corticosterone determinations
Plasma aldosterone was measured by RIA as described previously
(18). Plasma was extracted by the solid phase procedure using Amprep
cartridges (Amersham Canada). Then, the RIA was performed with a
specific antibody (Immunocorp). Plasma corticosterone was measured
directly with a RIA kit (Immunocorp).
Statistical analysis
The results of plasma determinations and enzymatic activities
were compared by two-factor ANOVA and Dunnett’s test where applicable.
For mRNA determination, Kruskal-Wallis one-way ANOVA on ranks was
followed by the Newman-Keuls test using the computerized Sigmastat
Statistical Analysis System (Jendel Corp., Corte Madera, CA). The data
are expressed as the mean and SEM of at least three
experiments (numbers given in parentheses). A probability
level of at least P < 0.05 was considered
significantly different.
Drugs and chemicals
All salts used in these experiments were of analytical grade and
obtained from Fisher Scientific (Montreal, Canada). Steroids were
obtained from Sigma (St. Louis, MO). All products used for RNA analysis
were purchased from Amresco (Intermedico, Markham, Canada).
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Results
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Plasma aldosterone and corticosterone levels during pregnancy
As shown in Table 1
, gestation was accompanied by a
significant increase in plasma aldosterone, which was apparent at 14
days gestation (2.53 ± 0.34 vs. 1.80 ± 0.20
pmol/ml in nonpregnant rats). Plasma aldosterone reached a maximal
value at 22 days gestation (3.41 ± 0.44 pmol/ml). This was
followed by a significant decrease at 18–36 h postpartum (0.60 ±
0.09 pmol/ml). Plasma corticosterone significantly decreased as early
as 7 days gestation (0.33 ± 0.05 vs. 0.61 ± 0.04
nmol/ml for nonpregnant rats). This reduction of plasma corticosterone
was maintained throughout gestation and early postpartum (14 days
gestation, 0.43 ± 0.03; 22 days gestation, 0.46 ± 0.03;
18–36 h postpartum, 0.35 ± 0.04 nmol/ml).
Activity of steroidogenic enzymes involved in aldosterone
biosynthesis
P450aldo activity was increased significantly at 14 days gestation
(2.41 ± 0.66 vs. 1.33 ± 0.16 pmol/capsule·min
aldosterone formed in nonpregnant rats (Table 2). This
increase was maintained until 18–36 h postpartum (3.59 ± 0.11
and 2.45 ± 0.34 pmol/caps·min of aldosterone formed in 22-day
pregnant and 18–36 h postpartum rats, respectively) and returned to
the prepregnancy level by 2 weeks after parturition (1.51 ± 0.18
pmol/capsule·min). The activities of P45011ß and P450scc were not
modified in the zona glomerulosa during rat gestation (data not
shown).
Expression of steroidogenic enzymes involved in aldosterone
biosynthesis
Blot hybridization patterns of P450scc revealed a clear single
band at 1.9 kb in the rat adrenal zona glomerulosa (Fig. 1) and rat adrenal zona fasciculata-reticularis (Fig. 2). The upper panel presents an autoradiogram
of a representative experiment. Quantitation data for mRNA signals were
standardized with a 32P-labeled ribosomal probe. The
relative intensities of P450scc mRNA from three separate experiments
are illustrated in the lower panel. Figure 1 demonstrates
that the quantity of this mRNA was not modified with gestation in the
zona glomerulosa. It was increased at 18–36 h postpartum in the zona
fasciculata-reticularis, but this change did not reach statistical
significance. P45011ß expression was also measured in the same
preparations. No signal was observed in the zona glomerulosa. Figure 3 (upper panel) shows a single band at 2.7 kb
in the zona fasciculata-reticularis. No effect of gestation was
observed on P45011ß expression (lower panel). The level of
adrenal P450aldo mRNA in the zona glomerulosa was increased at 14 days
gestation, but reached statistical significance only at 22 days
gestation (Fig. 4). Parturition induced a significant
decrease in P450aldo mRNA levels, as observed at 18–36 h postpartum
(vs. 22 days gestation), dropping below nonpregnant values
(P < 0.05). This mRNA returned to control levels at 2
weeks postpartum. No signal was observed in the zona
fasciculata-reticularis.
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Figure 1. Relative levels of P450scc mRNA in the adrenal
zona glomerulosa of nonpregnant (N-P) rats; rats at 7 days (7D), 14
days (14D), and 22 days (22D) gestation; and rats 18–36 h (18h) and 2
weeks (2w) postpartum (PP). Northern blot analyses were performed on 25
µg total RNA. Upper panel, Autoradiogram of a
representative experiment. Lower panel, Mean ±
SEM of three separate experiments, after standardization
with the 18S ribosomal probe.
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Figure 2. Relative levels of P450scc mRNA in the adrenal
zona fasciculata-reticularis of nonpregnant (N-P) rats; rats at 7 days
(7D), 14 days (14D), and 22 days (22D) gestation; and rats 18–36 h
(18h) and 2 weeks (2w) postpartum (PP). Northern blot analyses were
performed on 25 µg total RNA. Upper panel,
Autoradiogram of a representative experiment. Lower
panel, Mean ± SEM of three separate
experiments, after standardization with the 18S ribosomal probe.
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Figure 3. Relative levels of P45011ß mRNA in the adrenal
zona fasciculata-reticularis of nonpregnant (N-P) rats; rats at 7 days
(7D), 14 days (14D), and 22 days (22D) gestation; and rats 18–36 h
(18h) and 2 weeks (2w) postpartum (PP). Northern blot analyses were
performed on 25 µg total RNA. Upper panel,
Autoradiogram of a representative experiment. Lower
panel, Mean ± SEM of three separate
experiments, after standardization with the 18S ribosomal probe.
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Figure 4. Relative levels of P450aldo mRNA in the adrenal
zona glomerulosa of nonpregnant (N-P) rats; rats at 7 days (7D), 14
days (14D), and 22 days (22D) gestation; and rats 18–36 h (18h) and 2
weeks (2w) postpartum (PP). Northern blot analyses were performed on 25
µg total RNA. Upper panel, Autoradiogram of a
representative experiment. Lower panel, Mean ±
SEM of three separate experiments, after standardization
with the 18S ribosomal probe. *, P < 0.05
vs. nonpregnant values. P < 0.05,
22D vs. 7D. P < 0.05 22D
vs. 18hPP. P < 0.05 18hPP
vs. 2wPP.
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Discussion
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We report that P450aldo activity and expression follow the same
pattern as plasma aldosterone concentrations, which increased on the
14th day of gestation and decreased significantly 18–36 h after
parturition. Our results also show that P450scc do not appear to be
rate-limiting enzymes for aldosterone synthesis during pregnancy. The
11ß-hydroxylation of deoxycorticosterone could be produced by
P450aldo in zona glomerulosa and by P45011ß in zona
fasciculata-reticularis. The 11ß-hydroxylase activity in the zona
glomerulosa was not changed by pregnancy. Thus, we clearly demonstrated
for the first time that the increase in plasma aldosterone levels
during pregnancy is associated with augmented aldosterone synthase
activity and P450aldo mRNA levels in the zona glomerulosa. This
zone-specific effect is not surprising because aldosterone is
synthesized exclusively in the zona glomerulosa. At 18–36 h
postpartum, there is a significant decrease in the expression of
P450aldo and plasma aldosterone. However, the activity of P450aldo is
still higher than that in nonpregnant rat. This could be interpreted as
indicating that, despite the reduced transcription of P450aldo, after
parturition the remaining enzymatic protein was not yet degraded.
However, the increase in plasma ANP levels during this postpartum
period (22) is responsible for the decreased secretion of aldosterone,
although the enzyme is still present with significantly elevated
activity. This interpretation remains to be confirmed experimentally.
P45011ß activity and mRNA were not affected by gestation in the zona
fasciculata-reticularis. However, we observed reduced plasma
corticosterone levels during pregnancy. Published data on
corticosterone levels during rat gestation are controversial. Whipp
et al. (23) found no difference between proestrus, estrus,
and 18 days gestation, but the corticosterone level was lower in
diestrous rats. Our results are consistent with those of Churchill
et al. (24) and Garland et al. (2), who reported
a decrease in corticosterone at 22 days gestation. Schneider and Mulrow
(1) have also shown that adrenals from pregnant rats, when incubated
in vitro, produce less corticosterone than those from
nonpregnant rats. The discrepancy between the results of Whipp et
al. (23) and those of other groups could be explained by the time
of gestation (18 days instead of 22 days gestation) and the choice of
nonpregnant group (diestrus, proestrus, or estrus).
It seems that P450aldo was the most important enzyme in the regulation
of aldosterone synthesis. Indeed, many groups have demonstrated that
low sodium or high potassium intake induces modification of this
enzyme, whereas P45011ß remains unchanged (25, 26). Malee and Mellon
(27) found that pregnant rats had P450aldo and P45011ß mRNA in
essentially the same ratio as that in nonpregnant rat intact adrenals
(P450aldo/P45011ß mRNA, 1:10). In this study, we used two different
oligonucleotides that are specific for each mRNA, and when we compared
ratios for P450aldo in zona glomerulosa and P45011ß in zona
fasciculata-reticularis, we had a greater ratio for P450aldo/P45011ß
mRNA in pregnant than in nonpregnant rats. This difference between our
results and those of Malee and Mellon (27) could be explained by the
different techniques used; their analysis was performed using a
ribonuclease protection assay in whole intact adrenal, whereas we
employed specific oligonucleotides for each mRNA in their specific
zones of expression, and we compared these two zones.
In summary, we have demonstrated for the first time that elevated
plasma aldosterone levels during pregnancy are attributed to increased
activity and expression of P450aldo in the adrenal.
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Acknowledgments
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The authors thank Mrs. Lyne Ducharme for her technical
assistance.
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Footnotes
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1 This work was supported by a grant from the Heart and Stroke
Foundation of Quebec and the Fonds de la Recherche en Santé du
Québec. 
2 Scholar of the Heart and Stroke Foundation of Canada.
Received December 5, 1996.
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Top
Abstract
Introduction
