From the
Dahl salt-sensitive (S) and salt-resistant (R) rats are widely
used to study genetic determinants of salt-sensitive hypertension.
Differences in blood pressure under a high sodium diet in these two
strains may be due to differences in the synthesis of
18-OH-11-deoxycorticosterone (18-OH DOC). This difference in 18-OH-DOC
synthesis is due to mutations in the Dahl R rat's gene for
P450c11
The etiologies of essential hypertension are unknown, but they
include both environmental and genetic factors. At least one form of
salt-dependent hypertension is due to a genetic lesion in the gene for
11
The Dahl salt-sensitive (S) rat is the
most widely studied genetic model of salt-sensitive hypertension. In
the Dahl S strain, supplemental dietary sodium increases blood
pressure, but in the Dahl salt-resistant (R) strain, supplemental
dietary sodium has little effect on blood pressure(12) .
Molecular variants in the coding sequence of P450c11
In the Dahl S rat, plasma
levels of aldosterone are lower than in the Dahl R
rat(15, 16, 17, 18) , and, therefore, it
has been assumed that genetically determined alterations in aldosterone
biosynthesis do not contribute to the strain differences in blood
pressure. The lower aldosterone concentrations in Dahl S versus Dahl R rats have been proposed to be a physiologic consequence of
the lower renin concentrations in Dahl S rats(19) . However, in
Dahl S rats, the ratio of aldosterone/renin in the adrenals and in the
plasma is greater than in Dahl R rats(18, 20) . This
raises the possibility that in Dahl S and Dahl R rats, differences in
aldosterone biosynthesis may involve something more than just
differences in plasma renin activity. Although the P450c11
Both Dahl S and
Dahl R rats given a normal diet produce much more P450c11
It is well established that in Dahl S rats, plasma and
adrenal concentrations of both renin and aldosterone are lower than in
Dahl R rats(18, 20) . This would suggest that disordered
aldosterone production is not an important determinant of the greater
blood pressure in S rats versus R rats. It has been proposed
that the decreased concentrations of aldosterone in Dahl S rats versus R rats are a physiologic consequence of the decreased
renin concentrations(19) . However, in Dahl S rats, the
aldosterone/renin ratios in the plasma and in adrenals are greater than
in Dahl R rats. This raises the possibility that reduced aldosterone
concentrations in Dahl S rats are not just a consequence of reduced
renin concentrations.
In the current study, we have found that the
amount of adrenal P450c11AS mRNA is similar in S rats versus R
rats and that in both strains, NaCl depletion increases P450c11AS mRNA
and NaCl loading decreases P450c11AS mRNA to the same extent. Thus, the
reduced plasma concentrations of aldosterone in S rats versus R rats cannot be readily attributed to physiologic reductions in
P450c11AS gene expression caused by reduced plasma concentrations of
renin. The finding that P450c11AS mRNA levels are similar in Dahl S and
R rats despite the fact that plasma and adrenal renin levels are known
to be reduced in the S strain suggests that Dahl S and R rats may
differ with respect to the transcriptional regulation and or message
stability of P450c11AS. Enhanced transcription or message stability of
P450c11AS in Dahl S rats versus R rats could account for
similar levels of P450c11AS message despite reduced renin levels in the
S strain. This would serve to explain the greater aldosterone/renin
ratios that have been reported in Dahl S rats versus R rats.
Given that P450c11AS mRNA levels are similar in Dahl S and R rats,
how does one account for the lower plasma levels of aldosterone
reported in the S strain? In transfection studies, we found that the
inherent enzymatic activity of aldosterone synthase is lower in the S
rat versus the R rat, owing to a lower apparent V
The
P450c11AS cDNA sequence of the Dahl S rat is identical to the P450c11AS
cDNA and genomic sequence published for a Sprague-Dawley rat (21, 30),
whereas the P450c11AS sequence of the Dahl R rat differs by seven
nucleotides, two of which alter the predicted protein sequence of
aldosterone synthase. This is similar to what was found for the
P450c11
The observation
of specific amino acid residues associated with substantial differences
in aldosterone synthase activity in Dahl S and R rats recently prompted
us to investigate whether similar variations could alter the enzymatic
activity of human P450c11AS. When the mutations found in the Dahl R
P450c11AS were made at the corresponding residues in the human
P450c11AS, they resulted in 50-80% more aldosterone production
than the wild-type P450c11AS cDNA(34) . Thus, identification of
variants affecting aldosterone synthase activity in rats could be
relevant to the molecular control of aldosterone synthase in humans.
This raises the possibility that in humans, naturally occurring
variation in the corresponding regions of P450c11AS might contribute to
inherited variation in aldosterone production.
We thank Dr. Walter L. Miller and members of his
laboratory for providing the data on the activity of the human
P450c11AS synthetic mutations prior to publication. We also thank
Susanna Bair for performing the kinetic assays and for overall
excellent technical assistance.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(11
-hydroxylase), an adrenal enzyme involved in the
synthesis of both corticosterone and 18-OH DOC from
11-deoxycorticosterone. Aldosterone/renin ratios in plasma and in the
adrenals are greater in Dahl S than R rats, suggesting an altered
physiologic relationship between the renin-angiotensin and aldosterone
systems between these strains. We demonstrate that the mRNA for
P450c11AS, (aldosterone synthase), an enzyme required for aldosterone
synthesis, is identical in the Dahl S rat and in normotensive
Sprague-Dawley rats, but that P450c11AS mRNA from the Dahl R rat
contains 7 mutations that result in two amino acid substitutions. These
two changes result in a form of P450c11AS that has a greater apparent V
and lower apparent K
, resulting in an enzyme that catalyzes
the conversion of 11-deoxycorticosterone to aldosterone at a greater
rate in Dahl R rats than the P450c11AS in Dahl S rats or Sprague-Dawley
rats. Although plasma and adrenal renin are lower in Dahl S versus R rats, the regulation of P450c11AS mRNA expression in rats fed a
low and high salt diet are identical in these strains. The current
findings may explain both the reduced aldosterone concentrations and
increased aldosterone/renin ratios previously reported in the Dahl S versus Dahl R rat.
-hydroxylase, P450c11
, and aldosterone synthase,
P450c11AS(1) . Both the rat and human forms of this P450c11
encode a mitochondrial protein found in the adrenal
fasciculata/reticularis that synthesizes corticosterone from
11-deoxycorticosterone (DOC)
(
)and cortisol from
11-deoxycortisol, while the rat and human forms of P450c11AS encode a
mitochondrial protein found in the glomerulosa that synthesizes
aldosterone from
corticosterone(2, 3, 4, 5, 6, 7, 8) .
In patients with glucocorticoid-remediable aldosteronism, fusion of the
regulatory regions of the P450c11
gene to the coding region of the
P450c11AS gene results in disordered steroid synthesis,
hyperaldosteronism, and hypertension(9) . Several other rare
forms of secondary hypertension also result from elevated
concentrations of mineralocorticoids(10, 11) . This
raises the possibility that alterations in either P450c11
or
P450c11AS may contribute to the pathogenesis of other forms of
salt-sensitive hypertension.
have recently
been identified that may contribute to the differences in blood
pressure between the S and R strains(13, 14) . The
P450c11
gene of the Dahl R rat differs from that of 12 commonly
used rat strains, including the Dahl S rat, and contains missense
mutations that alter 11
-hydroxylase activity and decrease the
production of 18-OH-DOC. Moreover, in genetic studies, these mutations
have been found to cosegregate with reduced adrenal capacity to
synthesize 18-OH-DOC and resistance to the hypertensinogenic effects of
NaCl. These observations suggest that the robust salt resistance of the
Dahl R rat relative to the Dahl S rat may be due in part to molecular
variants in P450c11
and alterations in the activity of
11
-hydroxylase. However, in rats as in other mammals, the
P450c11
gene is tightly linked to P450c11AS, and therefore, blood
pressure effects that cosegregate with P450c11
could also be
emanating from variants in P450c11AS.
gene
has been well characterized in Dahl S and R rats, little is known about
the structure or function of P450c11AS in this model. In the current
studies in Dahl S and R rats, we cloned, sequenced, and expressed
P450c11AS cDNAs from these strains and studied the regulation of
P450c11
and P450c11AS mRNAs. Our results suggest that strain
differences in the structure and regulation of P450c11AS may be related
to the strain differences in aldosterone concentrations and
aldosterone/renin ratios.
Cloning and Sequencing P450c11AS cDNAs from
Dahl-resistant and Dahl-sensitive Rat Adrenals
Reverse
transcription and polymerase chain reaction were used to prepare and
clone P450c11AS cDNAs from the adrenals of three Dahl R and three Dahl
S rats. Rats were treated with furosemide (2 mg for 2 days) to induce a
sodium diuresis and stimulate P450c11AS synthesis, and with
dexamethasone (20 µg/animal) to suppress P450c11 mRNA
synthesis(3) . RNA isolated by guanidinium isothiocyanate
solubilization and CsCl gradient ultracentrifugation was reverse
transcribed using with the Superscript cDNA Synthesis Kit (Life
Technologies Inc.). Our polymerase chain reaction amplification of cDNA
used two sets of P450c11AS-specific primers so that the amplified
products contained overlapping sequences. The first polymerase chain
reaction amplified exons 1-4 and used a 5` primer that included
the first 6 amino acids of exon 1 (5`-GGATGGCAATGGCTCTCAGGGTGACA-3`)
and a 3` primer that spanned the first 6 amino acids of exon 5
(5`-CTTCCAGATACATCTGTTGG-3`). An EcoRI cloning site was added
at the 5` end of the 5` primer. The 3` primer has 3`-terminal
mismatches for P450c11
and is specific for P450c11AS. The second
polymerase chain reaction amplified exon 4-9, and used a 5`
primer specific for P450c11AS (nucleotides 651-672(21) )
(5`-CCCTGGTAGCCTGAAGTTCATC-3`) and a 3` primer (nucleotides
1526-1547 (21)) that distinguished P450c11
from P450c11AS
(5`-AGAAGTAGGCCGGTGGACTGAT-3`). An XbaI cloning site was added
to the 5` end of the 3` primer. Twenty nanograms of cDNA were amplified
for 30 cycles using the following conditions: 94 °C for 30 s, 55
°C for 30 s, 72 °C for 1 min. Amplified fragments were digested
with XbaI and HindIII, purified by agarose gel
electrophoresis, ligated, and cloned into M13 and sequenced by dideoxy
sequencing (Sequenase, U. S. Biochemical Corp.). All but the first 18
nucleotides of the coding sequence could be compared.
Expression of P450c11AS cDNAs after Transfection into
MA-10 Cells
Full-length P450c11AS cDNAs from both Dahl S and
Dahl R rats were cloned into pECE (22) and were transfected into
mouse Leydig MA-10 cells by calcium phosphate precipitation. After 48
h, the cells were incubated with varying concentrations of cold
corticosterone for 24 h. Enzymatic activity was assayed by determining
the 0-48 h time courses for aldosterone production using
10M 11-deoxycorticosterone in the
incubation medium or by determining aldosterone production using
10
-10
M
11-deoxycorticosterone in the medium for 24 h. Medium was analyzed for
aldosterone by RIA (ICN Biochemicals, Costa Mesa, CA).
Animals
Dahl salt-resistant (SR/Jr strain,
hereafter referred to as R rats) and Dahl salt-sensitive rats (SS/Jr,
hereafter referred to as S rats) were obtained from
Harlan-Sprague-Dawley Inc., prior to the occurrence of genetic
contamination in the Dahl strain (23). They were divided into four
groups and received one of four regimens for 7 days(3) . Control
animals were fed rat chow and water ad libitum.
Dexamethasone-treated animals received daily subcutaneous injections of
20 µg of dexamethasone in 100 µl of normal saline and were fed
rat chow and water ad libitum. Sodium-restricted animals (low
salt) received 2 mg of furosemide subcutaneously for 2 days and were
fed sodium-deficient rat chow (background sodium, 0.0033%) (Teklad,
Madison, WI) and water ad libitum. Sodium-replete animals
(high salt) received 2% NaCl in their drinking water and rat chow ad libitum. After 7-days of each treatment, the rats were
anesthetized with metaphane and killed by decapitation at 1300 h. Pairs
of adrenals were frozen separately in liquid nitrogen.
Preparation of RNA
-RNA was extracted from
adrenals by guanidinium isothiocyanate homogenization, purified by
cesium chloride ultracentrifugation as described previously(3) ,
and quantitated by determining the optical density at 260 and 280 nm.
Preparation of RNA Probes
Three different cRNA
probes were prepared by transcription of various cDNA fragments cloned
in pKS (Stratagene, La Jolla, CA) to analyze the abundance of
P450c11 and P450c11AS mRNAs in the adrenal. The P450c11 probe was
a 280-base RNA probe containing 219-bases of rat P450c11
cDNA
(corresponding to nucleotides 213-432(24) ) that
hybridizes to both P450c11
and to P450c11AS mRNAs. When hybridized
to normal rat P450c11
mRNA, a fragment of 219 bases is protected,
and when hybridized to normal rat P450c11AS mRNA, a fragment of 192
bases is protected(3) . A 290-base RNA probe containing
185-bases of rat P450c11AS cDNA (corresponding to bases
809-994(5, 24) ). A 260-base RNA probe containing
a 155-base pair rat P450c11
cDNA fragment (corresponding to bases
839-994(5, 24) ). To analyze P450scc mRNA, a
241-base pair EcoRI-PvuII fragment of rat P450scc
cDNA(25, 26) was cloned in pKS, generating a 296-base
RNA probe that protects a 233-base RNA fragment. As a control, a
195-base rat actin RNA probe containing a 149-base pair rat
actin
cDNA fragment (corresponding to nucleotides 2066-2216 of rat
cytoplasmic
-actin(27) ) was synthesized by reverse
transcription polymerase chain reaction of rat liver RNA(5) .
RNA probes were synthesized as described previously (28).
RNA Analysis
RNA was analyzed by RNase protection
assays (3, 5). One microgram of RNA from the adrenal was precipitated
with 500,000 cpm of P-labeled cRNA probe and was
resuspended in 20 µl of hybridization buffer containing 80%
formamide, 400 mM NaCl, 40 mM Pipes, pH 6.4, 1 mM EDTA. Samples were heated to 95 °C for 5 min and incubated at
42 °C overnight. Samples were then diluted 10-fold with buffer
containing 300 mM NaCl, 10 mM Tris-Cl, pH 7.9, 5
mM EDTA, and 20 µg/ml RNase A. Samples were incubated for
30 min at 42 °C, extracted once with phenol/chloroform, and
precipitated with ethanol. RNase-resistant double-stranded fragments
were separated on 6% polyacrylamide, 7.5 M urea/sequencing
gels using
P-labeled MspI-digested pBR322 DNA as
molecular weight markers. The gels were dried onto filter paper and
exposed to x-ray film under an intensifying screen at -70 °C.
Steroid Analysis
Medium was saved from
transfection assays and was analyzed for aldosterone by RIA using a
commercially available kit (ICN Biochemicals, Costa Mesa, CA).
Cloning P450c11AS cDNA from Dahl S and Dahl R
Rats
The sequence of the P450c11AS cDNA (from nucleotides 1 to
1547) from the Dahl S rat was identical to that of the outbred
Sprague-Dawley rat and thus predicted identical amino acid sequences.
However, the sequence of the P450c11AS cDNA from the Dahl R rat
contained seven nucleotide differences. Five of these seven differences
were silent mutations, but two changed the encoded amino acid sequence
of P450c11AS (): Glu 136 Asp and Gln 251
Arg.
Both of these substitutions in the Dahl R P450c11AS sequence generate
the same amino acids found at the corresponding residue of the
P450c11
gene.
Expression of P450c11AS cDNAs and Analysis of Their
Activities
We cloned P450c11AS cDNAs from the Dahl S and Dahl R
rats in the SV40 eukaryotic expression vector, pECE(22) . These
plasmids were then transfected into mouse Leydig MA-10 cells, which do
not normally express P450c11AS, but which contain the mitochondrial
electron transfer proteins adrenodoxin and adrenodoxin reductase that
are needed for P450c11AS activity. P450c11AS from the Dahl R and Dahl S
rats synthesized aldosterone from corticosterone (Fig. 1).
However, the amounts of aldosterone synthesized were radically
different. The Dahl R P450c11AS synthesized >1000 times more
aldosterone than did the Dahl S P450c11AS. This was not due to
differences in the transfection efficiencies of the plasmids containing
each cDNA, since all were co-transfected with a probe for
RSV-luciferase and corrected for transfection efficiency. This
difference was also not due to differential expression and stability of
the different forms of P450c11AS mRNA in the transfected MA-10 cells,
as RNase protection assays showed that cells transfected with both cDNA
plasmids expressed P450c11AS mRNA to the same degree (not shown). These
data suggest that the proteins encoded by these mRNAs are inherently
different enzymatically.
Figure 1:
RIA
analysis of aldosterone secreted from MA-10 cells transfected with
pECE-P450c11AS expression vectors. One microgram of
pECE-P450c11AS or pECE-P450c11AS
was
co-transfected into MA-10 cells with 0.5 µg of RSV-luciferase as an
internal control for transfection efficiency. Forty eight hours after
transfection, cells were incubated for 24 hr with 10
M 11-deoxycorticosterone. Aldosterone from the medium
was analyzed in duplicate, and was corrected for transfection
efficiency. Data from the P450c11AS
cDNA expression vector
are shown in the black bar. Control cells transfected with the pECE
vector alone had aldosterone concentrations of less than 0.02 pg/ml.
Results are ± S.E. from three separate transfections, each done
in triplicate.
To characterize the enzymatic activity of
each of these forms of P450c11AS, we incubated transfected MA-10 cells
with varying concentrations of 11-deoxycorticosterone for various
periods of time (Fig. 2). All samples were co-transfected with a
vector for RSV-luciferase to correct for transfection efficiency. At
all DOC concentrations and at all times, P450c11AS from Dahl R rats
converted more DOC to aldosterone than did P450c11AS from Dahl S rats.
Lineweaver-Burk analysis of the enzymatic conversion of DOC to
aldosterone indicated an apparent V of 0.0044 pg
of aldosterone/ml/min for the Dahl S P450c11AS and an apparent V
of 4.57 pg of aldosterone/ml/min for the Dahl
R P450c11AS. Similarly, Lineweaver-Burk analysis of the enzymatic
conversion of DOC to aldosterone indicated an apparent K
of 19.26 µM for the Dahl S
enzyme and an apparent K
of 2.15
µM for the Dahl R P450c11AS. Thus, the P450c11AS from the
Dahl R rat works faster and at lower substrate concentrations than does
the Dahl S P450c11AS enzyme.
Figure 2:
RIA analysis of aldosterone. MA-10 cells
transfected with 1 µg of pECE-P450c11AS expression vectors and 0.5
µg of RSV-luciferase were incubated with varying concentrations of
11-deoxycorticosterone (A and B) or with
10M 11-deoxycorticosterone for varying
times (C). Experiments were performed as described in the
legend to Fig. 1. Data from the P450c11AS
cDNA expression
vector are in panelsA and D and are shown
as closedcircles in panelC; data
from the P450c11AS
cDNA expression vector are in panelsB and E and are shown as opencircles in panelC. Lineweaver-Burk
replots of the data from A and B are shown in D and E. Aldosterone from the medium was
analyzed in duplicate by RIA and was corrected for transfection
efficiency. Results are ± S.E. from three separate
transfections, each done in triplicate.
Detection of P450c11
We used a P450c11,
P450c11AS
, and P450c11AS
mRNAs with a Single Probe
RNA probe that detects both P450c11
and P450c11AS mRNAs, but
distinguishes them by size on polyacrylamide
gels(3, 5, 29) . The nucleotide sequences of the
cloned genes and cDNAs for P450c11
(24, 30) and
P450c11AS (21, 30) are known. We chose a region
corresponding to bases 213-432 (24) of P450c11
for
the probe (Fig. 3). The probe contains 219 bases of P450c11
sequences and 61 bases of vector sequences. Hybridization of this probe
to P450c11
mRNA from either Dahl salt-sensitive or -resistant rats
yields a protected fragment of 219 bases. (Mutation at base 379 in the
Dahl R P450c11
mRNA from a C
U does not result in an RNase
A-sensitive site.) In the region of DNA used as probe, there are eight
differences between the sequence of P450c11AS
and
P450c11
used as probe, and six differences between the sequence of
P450c11AS
and P450c11
used as probe (Fig. 3). However, only the differences at bases 405 and 429 (underlined in Fig. 3) result in a mismatched RNA hybrid
that is sensitive to RNase A digestion(3, 29) . Thus
P450c11AS mRNA from both normal Sprague-Dawley and from Dahl S rats
will protect a probe fragment of 192 bases (from base 213 to base 405).
In the Dahl R rat, there are mutations at bases 405 and 408 that change
the base to the corresponding base in P450c11
and thus do not
create mismatched base pairs. However, the P450c11AS sequence in both
the Dahl R and S rats differs from the sequence of P450c11
at base
429. This RNase A-sensitive mismatch results in the protection of a
fragment of 216 bases. This is not seen in the Dahl S rat, since the
smaller, 192 base fragment, lacking this region, is generated. Thus,
our RNase protection assay can distinguish the mRNAs for P450c11AS from
Dahl S and R rats.
Figure 3:
Alignments of P450c11,
P450c11AS
(P450c11AS
) and
P450c11AS
(P450c11AS
) mRNA sequences
with the P450c11
cRNA used as probe in RNase protection assays.
The sequences are from nucleotide 213-432, as numbered by Nonaka et al. (24). Differences in eight nucleotides in the
P450c11AS
sequence and differences in six nucleotides in
the P450c11AS
sequence are noted below the c11
sequence. Nucleotides 405 and 408 in boldface show the
mutation in the P450c11AS
sequence. The lines under nucleotides 405 and 429 show the P450c11AS:P450c11
mismatched bases that are cleaved by RNase A digestion. The singleunderlined base 405 in P450c11AS
is sensitive
to RNase A digestion, resulting in a 192-nucleotide protected fragment.
The doubleunderlined base 429 in P450c11AS
is sensitive to RNase A digestion, resulting in a 216-nucleotide
protected fragment.
Regulation of P450c11
To determine if there was
differential regulation of the genes encoding the P450c11 and P450c11AS mRNAs in Dahl
sensitive and Dahl resistant Rats
and
P450c11AS mRNAs, we analyzed RNA isolated from Dahl S and Dahl R rats
given different dietary and hormonal treatments. Rats were given either
a high or low salt diet, which is known to alter P450c11AS mRNA
abundance(3, 31, 32) , or they were given
injections of dexamethasone, which is known to alter the abundance of
the mRNA for P450c11
but not for P450c11AS (3).
mRNA
than P450c11AS mRNA (Fig. 4). This is similar to what is seen in
normotensive Sprague-Dawley rats(3) . When given a low salt
diet, both strains of Dahl rats increase the abundance of P450c11AS
mRNA without affecting expression of P450c11
mRNA. The degree to
which P450c11AS mRNA is induced by a low salt diet is the same in the
Dahl S and Dahl R rats and is similar to that in normotensive
Sprague-Dawley rats(3) .
Figure 4:
RNase protection assay of RNA isolated
from adrenals of Dahl S and R rats given a low salt (LS) or
high salt (HS) diet, or normal rat chow (C). 2 µg
of adrenal RNA was combined with 1 10
cpm of
P-labeled P450c11
and actin cRNA probes, hybridized
overnight, digested with RNase A, and separated on 5% acrylamide. 7.5 M urea sequencing gels. Markers (M) are
P-labeled MspI pBR322 DNA fragments. The lane t contained 50 µg of tRNA and
P-labeled
probe.
As expected, there is no effect on
the expression of P450c11 mRNA in either the S or R rat in rats
given a high salt diet (Fig. 5). Using a greater amount of RNA
(20 µg rather than 2 µg) and using a probe specific for
P450c11AS mRNA (bases 809-994) (5) that does not contain any
differences between P450c11AS from the S and R rats, we could see that
in both Dahl S and R rats given a high salt diet, there is an
equivalent reduction in the abundance of P450c11AS mRNA (Fig. 5).
Figure 5:
RNase
protection assay of RNA isolated from adrenals of Dahl S and R rats
given a low or high salt diet, or injections of dexamethasone (Dex). 20 µg of adrenal RNA was combined with 1
10
cpm of
P-labeled P450c11AS and actin cRNA
probes, hybridized overnight, digested with RNase A, and separated on
5% acrylamide. 7.5 M urea sequencing gels. Lanes Dex (1) and Dex (3) are samples from both Dahl-sensitive and
-resistant rats treated with dexamethasone, but whose P450c11
mRNA
responses were different (see Fig. 6) Markers (M) are
P-labeled MspI pBR322 DNA fragments. The lane tRNA contained 50 µg of tRNA and
P-labeled
probe. Lane probes contains the P450c11AS and actin cRNA
probes.
Regulation of expression of both P450c11 and P450c11AS mRNAs by
dexamethasone was also studied ( Fig. 5and Fig. 6). In both
the S and R rat, like in Sprague-Dawley rats, dexamethasone selectively
decreases the expression of P450c11
mRNA, without affecting
expression of P450c11AS mRNA. In some cases, both Dahl S and Dahl R
rats are equally resistant to dexamethasone treatment, and P450c11
mRNA is unaffected (Fig. 6, Dex3, sensitive; Dex1, resistant). This
apparent lack of regulation by dexamethasone is similar in frequency
(about 1 in 3 rats) to that seen in Sprague-Dawley
rats(33) .
(
)This lack of P450c11
mRNA regulation was not indicative of abnormal regulation of P450c11AS
mRNA in either Dahl S or Dahl R rats (Fig. 5), since P450c11AS
mRNA from neither rat was affected by dexamethasone treatment. Thus,
there is no difference in the regulation of P450c11
or P450c11AS
mRNAs by dexamethasone between strains of Dahl rats, or between
normotensive Sprague-Dawley rats and Dahl rats.
Figure 6:
RNase
protection assay of RNA isolated from adrenals of Dahl S and R rats
given dexamethasone injections. 2 µg of adrenal RNA was analyzed
from each sample, as described in the legend to Fig. 4. Three animals
from each experimental group are shown. Samples from Dahl sensitive and
resistant rats given dexamethasone injections, dexamethasone 1 and 3
from each group, show different responses in P450c11 mRNA
regulation, and were also analyzed using a P450c11AS-specific probe
(see Fig. 5).
and a greater apparent K
of the Dahl S enzyme. This could contribute to the lower
plasma concentrations of aldosterone in S rats versus R rats
in the face of equivalent amounts of P450c11AS mRNA. Reduced
translational efficiency of P450c11AS mRNA from the Dahl S versus R rat could also contribute to lower aldosterone synthase activity
and lower plasma levels of aldosterone in the S strain versus R strain despite equivalent amounts of P450c11AS mRNA. The
transfection studies suggest that the S form of aldosterone synthase is
nearly 1000-fold less active than the R form of aldosterone synthase.
This 1000-fold difference in aldosterone synthase activity is far
greater than the modest 2-fold differences in plasma and adrenal
aldosterone levels reported between Dahl S and R
rats(18, 20) . This presumably reflects the fact that in
addition to the inherent enzymatic properties of aldosterone synthase,
other factors (e.g. substrate availability, translational
efficiency of P450c11AS mRNA, aldosterone clearance rate, etc.) also
influence circulating and tissue levels of aldosterone.
cDNA sequence of the Dahl S and R rats, as the
P450c11
cDNA sequence of the Dahl S rat was identical to that from
a Sprague-Dawley rat, and the sequence from the Dahl R rat was
different from both. However, in the case of P450c11AS, the nucleotide
changes result in amino acid changes that substantially increase enzymatic activity, while those for P450c11
result in changes
that slightly decrease enzymatic activity.
Table: Amino acid substitutions in P450c11AS from the
Dahl resistant rat
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.