(Hypertension. 1995;25:350-355.)
© 1995 American Heart Association, Inc.
Articles |
Testosterone Regulation of Renal 
2B-Adrenergic Receptor mRNA Levels
From the Department of Medicine, Creighton University Medical Center, Omaha, Neb.
Correspondence to Mark L. Johnson, PhD, Department of Medicine, Creighton University Medical Center, 601 N 30th St, Suite 6730, Omaha, NE 68131.
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Abstract |
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Abstract Androgens regulate blood pressure and renal
2-adrenergic receptors in a parallel fashion in the
spontaneously hypertensive rat (SHR). The present studies
investigated whether this regulation of renal
2B-adrenergic receptors occurs at the mRNA level. Male
and female SHR were gonadectomized at 4 weeks of age. The
gonadectomized rats were implanted with or without testosterone
propionate. Sham- gonadectomized rats served as controls. Total kidney
RNA was purified, and 2B-adrenergic receptor mRNA was
quantified with a reverse transcriptase–polymerase chain reaction
(RT-PCR) assay. The assay uses a mimic RNA added at known
concentrations to the sample RNA. The mimic was constructed from the
target sequence in the 2B-adrenergic receptor mRNA plus
a 20-bp insertion of a random nucleotide sequence. The amount of
2B-adrenergic receptor mRNA present in each sample
was obtained by determining the equivalence point between the amount of
RT-PCR product formed in the target band versus the mimic band, which
were resolved by gel electrophoresis. Intact males had more than two
times as much 2B-adrenergic receptor mRNA as intact
females. Castration of males reduced the male-female difference by more
than 60%. Ovariectomy slightly increased the
2B-adrenergic receptor mRNA level compared with that of
intact females. Treatment with testosterone elevated
2B-adrenergic receptor mRNA levels of gonadectomized
males and females to the level of intact males. The
2B-adrenergic receptor mRNA levels correlated remarkably
well with renal 2-adrenergic receptor density. We
conclude that testosterone regulates renal 2B-adrenergic
receptor gene expression at the mRNA level in the SHR.
Key Words: RNA, messenger • receptor, adrenergic, alpha • androgens • testosterone
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Renal
2-adrenergic
receptor (2-AR) density is increased in the
spontaneously hypertensive rat (SHR) and several other animal models of
genetic hypertension compared with their respective normotensive
control strains,1 2 3 4 5 6 7 suggesting that expression of renal
2-AR is controlled by genetic factors. The expression of
renal 2-AR is also regulated by dietary salt intake in
SHR, Dahl salt-sensitive hypertensive rats, and Sabra hypertensive
rats.1 2 3 4 Recently, we found that renal 2-AR
density is higher in male than in female SHR.7 Castration
of male SHR reduced the male-female difference in renal
2-AR density by 60%. In addition, testosterone
treatment raised renal 2-AR density of gonadectomized
male and female SHR to the level of intact male SHR. These data suggest
that 2-AR density is regulated by testosterone in SHR.
Furthermore, renal 2-AR density is regulated by both the
Y chromosome and the autosomes in SHR (M.L. Johnson, unpublished data,
1994). Interestingly, changes in renal 2-AR density by
these factors (genetic, dietary salt, and testosterone) are
consistently associated with parallel changes in blood
pressure,1 2 3 4 5 6 7 supporting our original hypothesis that
overexpression of renal 2-AR may play a role in the
pathogenesis of genetic hypertension.1
2-ARs are members of a receptor family that is
coupled to Gi proteins.8 Activation of
2-AR inhibits adenylate cyclase, increases
Na+-K+-ATPase activity, and regulates
K+, Na+, and
Ca2+ channels.8 9 10 11 2-AR
agonists enhance sodium reabsorption in renal tubules by promoting
Na+-H+ exchange and by activating
Na+-K+-ATPase.10 11 Because
the physiological effects of 2-ARs are dependent on
their density,12 SHR may have increased Na+
reabsorption through their increased renal 2-ARs. In
fact, increased Na+ reabsorption has been demonstrated in
renal brush border membranes in SHR.13
2-ARs are further classified into four subtypes:
2A-AR, 2B-AR, 2C-AR, and
2D-AR, according to their selectivity for different
ligands.14 The 2A-AR and
2B-AR subtypes are expressed in the rat kidney; 90% is
the 2B subtype.15 In addition,
[3H]rauwolscine binds with higher affinity to
2B-AR than 2A-AR.14
Therefore, most previous studies on renal 2-AR using
[3H]rauwolscine as the radioligand detected mainly the
2B-AR subtype.1 2 3 4 5 6 7 Thus, at least the
2B-AR subtype was increased in hypertensive rats in
these studies.2 3 4 5 7
The allosteric modulating site of renal 2-AR by
Na+ has been reported to be different between Sabra and
Dahl hypertensive rats and normotensive rats, suggesting a structural
difference in renal 2-AR between hypertensive and
normotensive rats.16 17 The basis for this suggestion is
that an alteration in the Na+ modulating site can be
induced by a point mutation in the 2-AR DNA coding
sequence.18 However, in a recent study,19
using an improved technique, we found that the Na+
modulating site of renal 2-AR is intact in both
Dahl hypertensive and normotensive rats. Thus, the basis for a
potential difference in the coding sequence of genomic DNA of
2-AR at the sodium modulating site between hypertensive
or normotensive rats is in doubt. Therefore, we have now focused on the
regulation of gene expression to better understand the mechanisms
whereby 2-AR is altered and how the alteration is
related to hypertension.
However, despite the potential importance of renal 2-AR
in sodium and water handling, little information is available
concerning the regulation of renal 2-AR concentration or
density. Changes in protein level, eg, 2-AR density,
could be driven at the level of gene expression and/or at the level of
protein turnover. Gene expression is regulated by differential rates of
transcription, processing of primary RNA transcripts, stabilization of
mRNA in the cytoplasm, and translation of mRNA into
protein.20 The purpose of the present studies was to
investigate whether renal 2B-AR is regulated by
testosterone at the mRNA level in SHR.
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Methods |
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Animals
Male and female SHR and Wistar-Kyoto rats at 4 weeks of age were purchased from Charles River Laboratories. The rats were maintained in 0.63x0.25x0.2 m3 cages (seven to eight per cage) at a temperature of 22°C, relative humidity of 30% to 50%, and constant 12-hour light/dark cycle. The rats were provided with tap water and Purina rat chow containing 1% NaCl. All animal protocols were approved by the University Animal Use and Care Committee.
Seven male and seven female SHR were gonadectomized under pentobarbital (50 mg/kg IP) anesthesia at 4 weeks of age. Seven male and eight female 4-week-old SHR were gonadectomized and implanted subcutaneously with 20 mm of silicone elastomer tubing packed with crystalline testosterone propionate and sealed with silicone elastomer medical adhesive, type A, as described before.7 Seven male and eight female SHR of the same age were sham operated and implanted with empty silicone elastomer tubing at the same time as the controls. All capsules were preincubated in phosphate-buffered saline for 24 hours before implantation. Capsules were changed every 4 weeks. Tail systolic pressure was measured indirectly with a physiograph (Narco Biosystems, Inc). The rats were killed at 20 weeks of age. Both kidneys were removed, quickly frozen with dry ice–methanol, and stored at -80°C. One kidney was used for [3H]rauwolscine binding assays7 ; the other was used for mRNA measurement. Methods of renal membrane preparation and saturation binding with [3H]rauwolscine were described previously.7
RNA Isolation
Total RNA from whole kidney was isolated with TriZOL reagent as
described by the supplier (Life Technologies). After purification, the
total RNA was treated with RNase-free DNase (amplification grade, Life
Technologies), inactivated with EDTA, and heated to 65°C for 10
minutes as described by the supplier. The RNA was ethanol-precipitated
and reconstituted in distilled, deionized water, and the concentration
was determined by measurement of the absorbance at 260 nm. All RNA
samples were stored frozen at -80°C.
Synthesis of Primer Oligonucleotides
The primers for constructing the 2B-AR mimic and
for quantifying 2B-AR mRNA levels by reverse
transcriptase–polymerase chain reaction (RT-PCR) were designed with
the computer program OLIGO from National Biosciences Inc.
The oligonucleotides were synthesized in our laboratory with a 391 DNA
synthesizer from Applied Biosystems Inc. After deprotection and
cleavage from the column, the oligonucleotides were purified with
Oligopurification cartridges (Cruachem) as recommended by the
manufacturer. Yields were determined by measurement of the optical
density at 260 nm.
Design and Synthesis of Mimic RNA Competitor
Fig 1 shows the strategy for mimic RNA
construction. Using the PC-based DNA sequence analysis package
DNASTAR (DNASTAR, Inc), we analyzed the reported cDNA
nucleotide sequences of 2A,
2B, and 2C21 22 to
identify regions of homology and nonhomology. Sequences from
nonhomologous regions were analyzed with the OLIGO program to construct left and right outside primers that will
produce a 335-bp sequence unique to 2B-AR in PCR
amplification. The internal primers were synthesized with an additional
20-bp "nonsense" sequence that is complementary to the sequence
added to the other half of the internal primer pair. These primers were
used in the initial construction of the 355-bp mimic RNA competitor.
The Table lists the left, right, and internal
oligonucleotides used. Separate PCR reactions were performed with the
left primer and its internal partner and the right primer and its
internal partner. The products were electrophoresed on 3% NuSieve
low-melting agarose (FMC) gels, and the desired band was cut out
and purified with Gelase (Epicentre Technologies). After purification,
the PCR products were combined, and a "zipping" PCR reaction was
performed with the left and right primers to produce a recombinant
competitor DNA (mimic DNA). After constructing the competitor
molecules, we subcloned the PCR products into the TA Cloning Vector
(Invitrogen, Inc). Recombinants were selected, and the orientation of
the insert relative to the T7 promoter contained in the vector was
determined with a PCR assay with the M13 reverse primer and the right
primer of cDNA target sequence. A clone with the proper orientation to
produce sense-strand RNA was selected when used as template for T7 RNA
polymerase in vitro transcription. Plasmid DNA was isolated from 50-mL
overnight cultures with Qiagen columns as described by the supplier
(Qiagen, Inc). PCR with the flanking M13 forward and reverse primers
was performed to produce a linear template (T7 mimic DNA) suitable for
RNA synthesis with T7 RNA polymerase. The PCR product was separated
from plasmid DNA by electrophoresis in a 3% NuSieve low-melting
agarose gel. The PCR product band was cut out of the gel and purified
with Gelase. This T7 mimic DNA was used as template for RNA synthesis
to produce a competitor RNA molecule (mimic RNA). [3H]CTP
was included in the mimic RNA synthesis to permit quantification of
product. After mimic RNA synthesis, the reaction was digested with
RNase-free DNase to destroy any mimic DNA, extracted with
phenol-CHCl3 followed by CHCl3, and
ethanol-precipitated. The amount of mimic RNA produced was quantified
by calculation of the molar incorporation of [3H]CTP into
trichloroacetic acid (TCA)-precipitable counts.
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Quantitative RT-PCR Assay
A constant amount of total RNA (500 ng) was used with each of
the four mimic RNA concentrations (range, 0.1 to 2 pg). cDNA synthesis
was conducted in a 20-µL volume for 15 minutes at 42°C, followed by
heat inactivation at 98°C for 5 minutes. Synthesis was primed with
the right 2B-AR primer. The final concentration of
reagents was 1x PCR buffer 2; 3.75 mmol/L MgCl2; 500
µmol/L dATP, dGTP, dCTP, and dTTP; 0.5 µmol/L right primer; 50 U
RT; and 20 U RNase inhibitor. Unless otherwise noted, all reagents were
purchased from Perkin-Elmer Corp (PE Xpress). Reaction tubes were
overlaid with 50 µL mineral oil to reduce evaporation. After reverse
transcription, 30 µL PCR reagent mixture was added to each tube. The
final PCR reaction (50-µL volume) contained the following components:
1x PCR buffer 2, 1.5 mmol/L MgCl2, 200 µmol/L of
each dNTP, 0.2 µmol/L of the left and right 2B-AR
primers, and 1.25 U Taq polymerase. Also, to provide for
later quantification, each reaction contained 5 µCi of
[32P]dCTP (3000 Ci/mmol, DuPont-NEN). The PCR portion was
performed in an automated thermal cycler TwinBlock System (Ericomp,
Inc) that was programmed as follows: 95°C for 5 minutes (initial
melt), followed by 28 cycles of 95°C for 1 minute (denature), 58°C
for 30 seconds (anneal), and 72°C for 1 minute (extend). The reaction
was completed with a final 5-minute extension at 72°C. Reaction
mixture (20 µL) was loaded onto a 3.0% Metaphor agarose gel (FMC)
and electrophoresed. The amount of product generated from the total RNA
and mimic RNA molecules was quantified with a PhosphorImager System
(Molecular Dynamics) and the IMAGEQUANT software. The log
of the ratio of RNA product to mimic product was plotted against the
log of mimic RNA molecules added to the reaction. A linear regression
analysis was performed, and the number of 2B-AR mRNA
molecules in the sample was determined by extrapolation to the
equivalence point (RNA product=mimic product, log ratio=0). Four
concentrations of mimic were used for each RNA sample. Negative control
reactions containing all the reagents except the RT were performed
before quantification to verify that contaminating genomic DNA had been
removed.
Data Analysis
Data are presented as mean±SEM. Differences among group
means were analyzed by ANOVA and the Newman-Keuls test. Linear
regression analysis was performed to correlate renal
2B-AR mRNA levels with 2-AR density and
blood pressure.
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Results |
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Several experiments were performed to optimize the conditions of the RT-PCR assay. We determined the linearity of the assay over a range of competitor concentrations and determined the optimal number of PCR cycles (not shown). The assay was maximally linear between 20 and 35 cycles in the range of concentrations measured in our total RNA samples. The amount of PCR product obtained after 28 cycles of amplification was identical for 15-, 30-, and 60-minute incubations with RT performed in the first step of the quantification (not shown). The essential step of removing contaminating genomic DNA from the total RNA samples was achieved by digestion of the kidney RNA samples with 15 U amplification grade DNase I per 100 µg of total RNA for 15 minutes, as recommended by the supplier.
Fig 2 shows representative PhosphorImager
images of RT-PCR electrophoresed in 3% Metaphor agarose gels. Clear
resolution of the mimic (355-bp upper band) and target
2B-AR mRNA (335-bp lower band) was obtained with our
method. As the concentration of mimic RNA competitor decreased, the
intensity of the target RNA increased. The counts in each band were
directly quantified by the PhosphorImager, and after correction for
background, the log of the ratio of target mRNA counts versus mimic RNA
counts was plotted against the log of the number of mimic RNA
molecules. Fig 2C shows representative graphs for female and
male groups. From the equivalence point, log ratio=0, the number of
2B-AR mRNA molecules was calculated and normalized to
the number of mRNA molecules per nanogram of total RNA. Total RNA from
each animal was analyzed similarly, and the mean and SEM for each group
were determined. Fig 3 shows the results of this
analysis. The renal 2B-AR mRNA level was
significantly higher in intact male than in intact female SHR.
Castration of male SHR reduced the 2B-AR mRNA level but
not to the level of intact females. Ovariectomy of female SHR tended to
increase the 2B-AR mRNA level, but the difference was
not significant (P>.05). Ovariectomized female SHR had
significantly (P<.01) lower 2B-AR mRNA
levels compared with intact male SHR. Testosterone treatment in
gonadectomized males and females raised the 2B-AR mRNA
level to that of intact males (P>.05).
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indicates intact rats; 
, gonadectomized
rats; 
, testosterone-treated rats; closed symbols,
males; and open symbols, females.
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Regression analysis of data among the six groups showed that the
2B-AR mRNA level (in terms of molecules per nanogram of
total RNA) correlated well with renal 2-AR density
(Bmax) and with blood pressure (BP) (Fig 4), giving the following equations:
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Fig 5 shows kidney weight, body weight, and the ratio of kidney weight to body weight. Body weight was significantly reduced by castration in male SHR (P<.01). Testosterone replacement restored the body weight compared with that of intact male SHR (P>.05). Female SHR gained weight after ovariectomy, and testosterone treatment increased their body weight further. Nevertheless, it was still lower than the body weight of all male groups, including intact, castrated, and testosterone-treated males. Kidney weight was significantly reduced by castration in male SHR. Although testosterone treatment increased kidney weight in castrated males, their kidney weight was still significantly lower than that of sham-operated male SHR (P<.05). Ovariectomy did not significantly increase kidney weight (P>.05). Interestingly, testosterone treatment increased kidney weight of ovariectomized females equivalent to that of testosterone-treated male castrates (P>.05) and greater than that of castrated males. Because of the disproportionate increase in kidney weight relative to body weight after testosterone treatment, female SHR treated with testosterone had the greatest ratio of kidney weight to body weight (P<.01) compared with any other group. Castrated males had the lowest ratio of kidney weight to body weight among all groups.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Several groups have reported the application of PCR to the measurement of mRNA levels.23 24 25 However, some questions and debate remain about the "true" quantitativeness of the method.26 We used a strategy based on the method of Diviacco et al27 to construct a mimic competitor for the
2B subtype. This approach involves the construction of a
competitor molecule that is longer than the native target sequence and
thereby resolved by gel electrophoresis. Many problems inherent in the
methods involving the construction of competitors engineered to contain
unique restriction enzyme sites (eg, heteroduplex formation) have been
circumvented by this method. We also synthesized mimic RNA that was
added at known concentrations to the total kidney RNA samples before
the reverse transcription step. This eliminates any problems with
variable efficiencies during reverse transcription that would otherwise
need to be carefully controlled and quantified.23
Using this improved PCR technique, we have demonstrated in the
present studies that 2B-AR mRNA levels are regulated
by testosterone in SHR. Our results show that intact male SHR had more
than two times as much 2B-AR mRNA as intact females.
After castration of male SHR, renal 2B-AR mRNA was
reduced by more than 60% of the male-female difference. Furthermore,
testosterone replacement in castrated males restored the level of
2B-AR mRNA, indicating that 2B-AR mRNA
levels are indeed regulated by testosterone. An interesting observation
of our study is that ovariectomized female SHR treated with
testosterone acquired the same level of 2B-AR mRNA as
intact male SHR. This finding suggests that there is no intrinsic
difference in the potentials to change 2B-AR mRNA levels
between male and female SHR in response to testosterone treatment.
In contrast to castration of male SHR, ovariectomy only slightly
increased 2B-AR mRNA levels, and the increase did not
reach a statistically significant level compared with intact female
SHR. Even though the elevation of the 2B-AR mRNA level
may represent an effect of estrogen, the magnitude of change
was far less impressive than that of testosterone. Therefore, androgens
rather than estrogens play the principal role in the regulation of
2B-AR mRNA, as is the case for the regulation of blood
pressure.
The present study confirms and extends our previous conclusions
based on results from [3H]rauwolscine binding
assays.7 The relation between mRNA level and the encoded
protein concentration is often not a simple one. In our study, however,
we found a linear relation between mRNA concentration and receptor
density. Linear regression analysis showed excellent correlation
between 2B-AR mRNA levels and the amount of the encoded
protein, the renal 2-AR, among sham-operated male and
female and gonadectomized SHR supplemented with or without
testosterone. This may be partly due to the power of the quantitative
RT-PCR technique to measure specific mRNA concentration accurately and
precisely.
It remains to be resolved whether the regulation of
2B-AR occurs at the level of transcription or mRNA
stabilization. However, most steroid-mediated effects appear to occur
at the level of regulating gene transcription28 mediated
through specific receptors. Our findings are consistent with the
results from studies on adipocytes showing that 2-AR is
regulated by androgens at the mRNA level.29 In this
system, the effect appears to be at the transcriptional level.
The findings of the present studies are consistent with the notion
that testosterone is an important physiological modulator of genes
involved in the development of hypertension in an animal
model.30 In humans, high blood levels of
testosterone have been reported in hypertensive
children.31 Deoxycorticosterone has been proposed to be
involved in androgen-induced hypertension because plasma
deoxycorticosterone concentrations are higher in testosterone-induced
hypertensive rats.32 The increased deoxycorticosterone
levels may be a result of decreased 11ß-hydroxylase cytochrome
P-450.33 The present studies provided another
alternative mechanism whereby testosterone regulates blood pressure,
ie, through regulation of renal 2-AR at the mRNA
level.
An interesting observation of the present study was that after treatment with testosterone, the kidneys of ovariectomized females became as large as those of castrated males treated with testosterone. Because their body weight was not increased as dramatically as their kidney weight, the ovariectomized females treated with testosterone had the highest ratio of kidney weight to body weight among the six groups. These observations emphasize the dramatic effect of the male sex hormone testosterone on the growth of the kidney in SHR.
In summary, renal 2B-AR gene expression is regulated by
testosterone at the mRNA level in SHR. The sex difference between males
and females in 2-AR density was also observed at the
mRNA level. However, there is no intrinsic difference in the potential
of renal 2B-AR mRNA to be regulated by testosterone
between male and female SHR. Nevertheless, the dramatic effects of
testosterone on renal 2B-AR mRNA, 2B-AR
density, and blood pressure7 are tightly associated and
are consistent with a causal relation.
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Acknowledgments |
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This work was supported by grant RO1 HL-30339 from the National Heart, Lung, and Blood Institute, National Institutes of Health, and by the Health Future Foundation. We thank Tonia Baldwin for her expert technical assistance.
Received July 14, 1994; first decision August 31, 1994; accepted November 2, 1994.
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M. Khalid, Y. Giudicelli, and J.-P. Dausse An Up-Regulation of Renal alpha 2A-Adrenoceptors Is Associated with Resistance to Salt-Induced Hypertension in Sabra Rats J. Pharmacol. Exp. Ther., December 1, 2001; 299(3): 928 - 933. [Abstract] [Full Text] [PDF]
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J. Alfie, G. D. Waisman, C. R. Galarza, M. I. Magi, F. Vasvari, L. M. Mayorga, and M. I. Camera Relationship Between Systemic Hemodynamics and Ambulatory Blood Pressure Level Are Sex Dependent Hypertension, December 1, 1995; 26(6): 1195 - 1199. [Abstract] [Full Text]
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