503

Immunohistochemical and Biochemical

Evidence for a Cardiovascular
Mineralocorticoid Receptor
Marc Lombes, Marie-Edith Oblin, Jean-Marie Gasc, Etienne Emile Baulieu,
Nicolette Farman, and Jean-Pierre Bonvalet

The presence of mineralocorticoid receptors (MRs) and their physicochemical characteristics were
investigated in the heart and blood vessels of rabbits. Immunohistochemical methods using the
monoclonal anti-idiotypic antibody H1OE, which interacts with the steroid binding domain of MRs,
Downloaded from http://circres.ahajournals.org/ by guest on October 11, 2017

revealed the presence of immunoreactive material in the heart and large blood vessels. In the heart, a
positive staining was observed in myocytes and endothelial cells of atria and ventricles. In vessels, MRs
were detected in the aorta and pulmonary artery. They were localized in endothelial and vascular smooth
muscle cells. No staining was present in the small vascular bed, arterioles, and capillaries. In all these
studies, the mineralocorticoid specificity of the staining was assessed by in situ competition experiments
with aldosterone and RU486, a glucocorticoid antagonist. The presence of MRs in the heart and vessels
was further demonstrated by specific aldosterone binding to one class of high affinity binding sites in the
cytosol of the adrenalectomized rabbit heart (Kd, 0.25 nM; maximum MR concentration, 15-20 fmol/mg
protein), whose mineralocorticoid specificity has been clearly established by competition studies.
Sedimentation gradient analyses revealed that the cardiovascular MR is an 8.5S hetero-oligomer that
includes the heat shock protein 90. The physicochemical characteristics of the cardiovascular MRs are
virtually identical to those of the renal MRs. Altogether, our results clearly demonstrate the presence of
MRs in the cardiovascular system. This supports the possibility of direct aldosterone actions in the heart
and blood vessels. (Circulation Research 1992;71:503-510)
KEY WoRDs * aldosterone * monoclonal antibodies * heart * blood vessels * rabbits

T he mineralocorticoid hormone aldosterone is in- is a mineralocorticoid-responsive organ since aldosterone

volved in the regulation of sodium balance and
body fluids and consequently participates in the
control of blood pressure (for review, see Reference 1).
Besides the action of aldosterone in the kidney, where it
regulates electrolyte transport in the distal parts of the
nephron, several studies argue in favor of an effect of this
hormone on cardiovascular functions through direct ac-
tions on the heart and vascular tissues. Aldosterone ac-
tions are thought to be mediated through binding to
specific intracellular receptors. Although several studies
have reported aldosterone binding in the heart23 and
arteries,4-8 many uncertainties remain concerning the
nature of aldosterone action on the cardiovascular system.
Experimental studies have reported that mineralocor-
ticoids act on arterial smooth muscle cells, leading to an
increased vasoreactivity to pressor agents.9'10 They may
modulate muscle tone and vessel elasticity and induce
variations of electrolyte composition of the arterial
wall.1"-' Additional studies have suggested that the heart
From INSERM U 33 (M.L., M.-E.O., E.E.B.), Labhormones,
Bicetre, France; INSERM U 36 (J.-M.G.), College de France, Paris;
and INSERM U 246 (N.F., J.-P.B.), UER Xavier Bichat, Paris.
Supported by Fondation Searle pour la Recherche pour l'Hy-
pertension (N. AD09OF039001-A).
Address for correspondence: M. Lombes, INSERM U 33,
Labhormones, Faculte de Medecine Paris-Sud, 80, rue du Gdneral
Leclerc, 94276 Bicetre Cedex, France.
Received July 17, 1991; accepted April 16, 1992.
stimulates Na+,K+-ATPase synthesis in rat cardiocytes'6
and may directly influence myocardial structure and
functions.'7,"8
Recently, the availability of a monoclonal anti-min-
eralocorticoid receptor antibody H1OE19 renders it pos-
sible to directly assess the presence of mineralocorticoid
receptors (MRs) in the heart and vessels. This anti-
idiotypic antibody H1OE, which interacts with the steroid
binding domain of MRs, has already been successfully
used to immunolocalize the receptor along the rabbit
nephron.20,21
The present study was undertaken to localize and
characterize MRs in the rabbit heart and vessels by both
biochemical and immunohistochemical methods. The
biochemical approach allows for the characterization of
the cardiovascular MR in terms of affinity and physical
structure and the quantification of its abundance among
various tissues. In addition, the distribution of MRs
among different cell populations can be determined by
immunohistochemistry. The present article demon-
strates the presence of MRs in the cardiovascular
system and provides a basis for further studies of the
mechanisms of aldosterone action in these tissues.
Materials and Methods
Buffers
The following buffers were used: TEG buffer, consist-
ing of 20 mM Tris-HCl, 1 mM EDTA, and 10% glycerol;
 504 Circulation Research Vol 71, No 3 September 1992
TEGW buffer, consisting of TEG buffer plus 20 mM
sodium tungstate; and TEW buffer, consisting of 20 mM
Tris-HCl, 1 mM EDTA, and 20 mM sodium tungstate at
pH 7.4. All buffers were adjusted to pH 7.4 (25C).
Zamboni fixative, consisting of 2% paraformaldehyde
(wt/vol), 15% saturated picric acid (vol/vol), and 85%
0.15 M sodium phosphate buffer at pH 7.4 (vol/vol), was
also used.
Chemicals
1,2-[3H]Aldosterone was purchased from Radio-
chemical Center, Amersham, UK. Unlabeled aldoste-
rone was from Sigma Chemical Co., St. Louis, Mo.
RU486 was obtained from Roussel Uclaf Laboratories,
Romainville, France. Spironolactone (SC9420) and
ZK91587 were from Searle Laboratories, Chicago, Ill.,
and NEN Research Products, Du Pont de Nemours,
France, respectively. To avoid steroid adsorption, the
steroid solutions prepared in ethanol were dried and
resuspended in 50% polyethylene glycol 300 prepared in
Downloaded from http://circres.ahajournals.org/ by guest on October 11, 2017
TEG buffer to reach a 5% final concentration of poly-
ethylene glycol 300 in the cytosol.
Antibodies
The following antibodies were used: H1OE4C9F, a
mouse immunoglobulin (Ig) Gi monoclonal antibody
that interacts with the steroid binding domain of the
mineralocorticoid receptor19; and 7C10, a mouse IgGI
monoclonal antibody directed against the rabbit heat
shock protein 90 (hsp90).22
Animals
Experiments were performed on New Zealand fe-
male rabbits weighing 1-2 kg. Some animals were
bilaterally adrenalectomized under anesthesia and
given 0.9% saline to drink ad libitum for 48 hours.
Normal or adrenalectomized animals were perfused in
situ with either 500 ml prewarmed Zamboni fixative for
immunohistochemical studies or 300 ml cold 0.9% sa-
line followed by 200 ml cold TEGW buffer for biochem-
ical analyses.
Immunohistochemical Technique
At the end of the perfusion, small pieces of different
tissues were postfixed in Zamboni fixative for 24 hours.
They were then washed several times with 70% (vol/vol)
ethanol, dehydrated in graded ethanol, cleared in
1-butanol, and embedded in Paraplast. Sections (7 ,Lm)
were cut, mounted on histological slides, and processed
for immunohistochemistry. A routine procedure of in-
direct immunostaining was used.23 After deparaffiniza-
tion and rehydration, sections were incubated with 1%
normal horse serum in phosphate-buffered saline. The
monoclonal anti-idiotypic antibody H1OE, a mouse
IgGl immunoglobulin, was incubated as diluted ascites
at a concentration of 1-5 ,gg/ml for 2 hours at room
temperature or overnight at 4C. A mouse IgGl, MOPC
31C (Sigma), was used as control. The mouse antibody
was followed by a horse biotinylated anti-mouse anti-
body (Vector Laboratories, Inc., Burlingame, Calif.),
and the avidin-biotin-peroxidase complex (ABC-Elite
from Vector Laboratories) was used as a detection
system. The slides were rinsed in phosphate-buffered
saline after each incubation step. The peroxidase activ-
ity was revealed by diaminobenzidine tetrahydrochlo-
ride (0.5 mg/ml) in the presence of 0.01% hydrogen
peroxide. Sections were dehydrated and mounted in
Canada balsam without counterstaining. In situ compe-
tition studies were performed by preincubating tissue
sections with 1-5 ,uM steroid (aldosterone or RU486) in
phosphate-buffered saline 30 minutes before incubation
with H1OE antibody together with the steroid.
Cytosolic Preparation
After in situ perfusion, various tissues or organs were
cut in small pieces and immediately frozen in liquid
nitrogen. Pieces were then ground in a mortar under
liquid nitrogen. TEGW buffer was added to the result-
ing powder (2-3 ml/g). As soon as the mixture had
thawed, it was vortexed at 0C and homogenized with a
Teflon-glass Potter apparatus at 4C. The crude homog-
enate was centrifuged at 105,000g for 60 minutes at 4C.
The resulting supernatant was immediately used or
frozen in liquid nitrogen with no change in the binding
activity or physicochemical properties of the MR.
Binding Studies
To determine aldosterone binding parameters at
equilibrium in the heart, increasing amounts of [3H]al-
dosterone (0.1-100 nM) were incubated with 50 ,l
cytosolic aliquots for 4 hours at 4C. Bound and un-
bound steroids were separated by dextran-charcoal
technique.24 The evolution of bound steroid as a func-
tion of unbound steroid was analyzed by a computerized
method.25 Competition studies were performed by incu-
bating 50 ,ul cytosolic heart aliquots for 4 hours at 4C
with 5 nM [3H]aldosterone in the absence or presence of
unlabeled steroids: aldosterone, SC9420, ZK91587, or
RU486. Unbound steroid was removed by dextran-
charcoal treatment. The results are expressed as the
percentage of [3H]aldosterone binding alone.
Density Gradient Analyses
Cytosolic samples were incubated with 5 nM [3H]al-
dosterone alone or in the presence of either 0.5 gLM
RU486 or the monoclonal antibody H1OE (1/100 diluted
ascites) for 4 hours at 4C. After dextran-charcoal
treatment, samples, generally 0.15 ml, were layered onto
the top of 15-40% glycerol gradients prepared in TEW
buffer. Gradients were prepared as six discontinous
layers and maintained at least 2 hours at 4C before use.
To determine the subunit composition of the MR, heart
cytosol was incubated with either a nonimmune mouse
IgGl or the monoclonal anti-hsp90 antibody, 7C10, for
4 hours at 4C and then layered onto the top of glycerol
gradients. Gradients were centrifuged for 18 hours at
4C at 50,000 rpm in a rotor (model SW60, Beckman
Instruments, Gagny, France). Two-drop fractions were
collected from the bottom of the tube after puncture,
and the radioactivity was counted. Myoglobin (2S re-
gion), bovine serum albumin (4.6S region), and aldolase
(7.9S region) were used as external standards in parallel
tubes. The quantification of MR content in various
rabbit tissues was achieved by counting the specific
radioactivity recovered in the 8S-9S region of the
gradient profile.
 Lombes et al Cardiovascular Mineralocorticoid Receptor 5Q5

m~~~~~
m

FIGURE 1. Immunodetection of mineralocorticoid receptors in rabbit aztriulm with the monoclonal anti-idiotypic antibody H1OE.
Serial sections of rabbit left atrium were either immunostained with H]1OE alone (panel A) or in the presence of aldosterone (panel
B) or stained with Masson's trichrome (panel C). Panel A: Immunostaining of the atrium with H1OE alone is localized over the
Downloaded from http://circres.ahajournals.org/ by guest on October 11, 2017

myocytes (m) and the endothelial cells (e). Note that some cells of the subendothelial layer (under the endothelial cell layer) seem
to be also positively stainzed. Panel B: The specificity of the immunodetection was achieved by mealns of in situ competition studies
(H] OE +aldosterone) as described in "'Materials and Methods. ' Immunostaining is grea tly reduced after preincubation ofthe tissue
section by 1 ,uM aldosterone. Panel C: Histological staining of the same atrial section shows m and e. The nulclei are strongly
stained. Magnification, x 216.

Miscellaneous 3B). These micrographs were taken from the section

Protein concentration was determined by Bradford's
method,26 and bovine serum albumin was used as a
standard. The position of standard proteins in the
gradient fractions was determined by Lowry's method.27
Results
Immunolocalization of MRs
Figure 1 examplifies the immunostaining obtained
with the monoclonal antibody H10E in the left atrium of
the rabbit heart. A strong labeling is visible in the
myocytes and the endothelial cells (Figure 1A). Within
the subendothelial connective layer of the left atrium,
some cells, presumably fibrocytes, also seem to be
labeled. When compared with a serial section stained
with Masson's trichrome (Figure 1C), it appears that the
immunostaining of myocytes is distributed over both
nuclear and cytoplasmic compartments. The specificity
of the immunoreactivity for the MR was established by
in situ competition studies in which preincubation with
aldosterone was performed before incubation with the
monoclonal anti-idiotypic antibody H10E. In this condi-
tion, the intensity of the staining is strongly reduced in
atrial myocytes as well as in the endothelial cells (Figure
1B). A murine monoclonal antibody MOPC31 unrelated
to MR, used as a control, did not give any significant
staining (data not shown).
The results obtained from rabbit aorta are presented
in Figure 2. Both endothelial cells and vascular smooth
muscle cells of the arterial wall are positive (Figure 2A).
As in the case of the left atrium, preincubation with
aldosterone significantly reduced the immunostaining
(Figure 2B). In contrast, RU486, a synthetic steroid that
does not bind MRs,2428 does not modify the pattern of
immunostaining (Figure 2C).
Figure 3 shows immunostaining observed in the left
ventricle (Figure 3A) and the pulmonary artery (Figure
used for photographs of Figures 1 and 2. This heart
section includes the left atrium (Figure 1A), left ventri-
cle (Figure 3A), the initial part of the ascending aorta
(Figure 2A), and the pulmonary artery (Figure 3B).
The fact that each part of this section was submitted to
strictly identical experimental procedures allows a di-
rect comparison of the relative intensity of the immu-
nostaining from one tissue to another. The distribution
of immunostaining in the ventricle is very similar to that
of the atrium. However, the intensity of labeling is
stronger in atrial myocytes than in ventricular myocytes.
On the other hand, the immunostaining seems to be
weaker in the aorta and the pulmonary artery. Although
quantitative interpretation of immunohistochemical
data is difficult, our results suggest that MR is relatively
more abundant in the heart, in particular the atrium,
than in large arteries.
Figure 4 illustrates the immunostaining observed in
the smaller vascular bed. In contrast to the aorta (Figure
2A) and the pulmonary artery (Figure 3B), only a weak
immunostaining was detected in smaller arteries such as
the carotid (Figure 4B), humeral, mesenteric, coronary,
and renal arteries (data not shown). No significant stain-
ing was apparent in the vena cava and portal vein (data
not shown). With respect to the arterioles and capillaries,
no MR immunoreactivity was detectable. Examples of
the absence of vascular staining are given for an intra-
myocardial capillary, contrasting with the strong labeling
of the myocytes (Figure 4A), and an intrarenal arteriole,
contrasting with the heavy immunostaining of a cortical
collecting duct (Figure 4C).
To study the effect of the endocrine status on cardio-
vascular MR expression, we examined heart and blood
vessel sections of normal or adrenalectomized rabbits.
In these conditions, we could not detect any difference
in the intensity of the immunostaining given by H1OE or
 506 Circulation Research Vol 71, No 3 September 1992

A B VI C
r_ -
_ v _ #_
,,-
Op

t r
-
-
-
C_

_ V. . - _I.-
t~
X

_ v
--- C
,>:ld
.1
,~

FIGURE 2. Immunodetection of mineralocorticoid receptors in the rabbit aorta. The sections include the endothelium (arrows),
the arterial wall with the vascular smooth muscle cells, and the tunica adventitia (bottom of each panel). Panel A: Monoclonal
Downloaded from http://circres.ahajournals.org/ by guest on October 11, 2017

antibody HfOE reveals immunoreactive material in the endothelial cells (indicated by the arrows) as well as in the vascular smooth
muscle cells of the aortic wall. Panel B: When aldosterone is applied to the tissue section together with the monoclonal antibody
HIOE, the immunostaining is clearly reduced. Panel C: In contrast, excess RU486 does not impair the immunoreactivity.
Magnification, x 344.
in the relative cellular distribution otf the labeling among one class of specific and high affinity binding sites, with
various tissues examined. a Kd value of 0.25 nM and a maximum number of sites
of 22.1 fmol/mg protein. A nonspecific binding was also
Biochemical Characterization of MIRs detected and corresponded to the /8 constant reported
Since a specific immunostaining was observed in the in the inset of Figure 5. The aldosterone binding sites
heart and the large blood vessel,s, we characterized detected in the cardiac cytosol clearly displayed a
[3H]aldosterone binding in the catrdiovascular system mineralocorticoid specificity, as demonstrated by com-
and studied the physicochemical piroperties of MRs in petition assays shown in Figure 6. Unlabeled aldoste-
these tissues. Biochemical analyses were performed in rone competed with [3H]aldosterone for binding to MRs
the cytosolic fractions of several tis,sues of adrenalecto- with 50% inhibition occurring for a concentration (IC,)
mized rabbits. We first measured akdosterone binding in of 0.5 nM consistent with the Kd value of 0.25 nM
the heart cytosol. Scatchard plolt representation of estimated from the Scatchard plot analysis. Two an-
[3H]aldosterone binding is presente d in Figure 5. Com- timineralocorticoid compounds, SC9420 and ZK91587,
puterized analysis of the experim' ental data revealed were potent competitors with IC50s of 3 and 10 nM,
respectively. In contrast, RU486, a synthetic steroid that
does not bind to MRs,2428 was unable to compete for
B e e aldosterone binding sites.
,- V *-Glycerol gradient centrifugation analyses of heart
cytosol labeled with tritiated aldosterone in the absence
- * or presence of RU486 or of the monoclonal antibody
HIOE were performed to determine the sedimentation
characteristics of the heart MR. As observed in Figure
1..
=- a 7A, aldosterone-MR complexes sedimented as an 8.5S
species (8.78+0.26S, n5). H1OE completely abolished
A.t
(Figure 7A), confirming the immunological recognition
1
of cardiac MRs by the monoclonal anti-idiotypic anti-
1

--
body

H1OE. profile of these receptors (data not

sedimentation
As expected, RU486 did not modify the

_/
.: -o
FIGURE 3. Immunostaining of the leftt ventricle and pulmo-
nary artery with monoclonal antibody FIIOE. Panel A: In the
left ventricle, a strong positive stainin) g is observed over the
myocytes (m). Magnification, x202.5. Panel B: In the pul-
monary artery, mineralocorticoid recep tors are detected in the
endothelial cells (e) and within the entire arterial wall.
Magnification, x 127.5.
..
shown). Like other steroid receptors, MR under its
.'A, large untransformed state is associated with a non-
DNA binding protein identified as the heat shock
protein of Mr -90,000 (hsp90).24 Therefore, the subunit
composition of the heart MR was studied using 7C10, a
monoclonal antibody that specifically recognized rabbit
hsp90.22 When aldosterone-labeled receptors were in-
cubated in the presence of 7C10, the peak of radioac-
tivity was clearly shifted to the liS region of the
gradient as illustrated in Figure 7B. This shift is ac-
 Lomb?s et al Cardiovascular Mineralocorticoid Receptor 507

A i C a
v
b%.t
c ~~~
! Am

FIGURE 4. Immunodetection of reactivity in the smaller arteries, arterioles, and capillaries. Panel A: A capillary (c) in the left
atrium is devoid of any immunoreactivity with H 1OE antibody. This contrasts with the strong surrounding positive staining of the
atrial myocytes (m). Panel B: A significant although weak immunostaining is detected in the carotid artery. The arrow e indicates
the position of the endothelial cells. Panel C: In the kidney, an interlobular artery (a) is negative as compared to the positivity of
a cortical collecting duct (ccd). Magnification, x344.
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counted for by the increase of the molecular weight
resulting from the binding of the antibody to the un-
transformed MR complex, indicating that the cardiac
MR is clearly a hetero-oligomer that includes hsp90.
The quantification of MR can be easily achieved by
counting the radioactivity recovered in the 8S-9S region
of the gradient. Using this method, MR concentration
in the heart cytosol was estimated to be 16.9 fmol/mg
protein, a figure close to that calculated from the
Scatchard plot analysis. We have also investigated the
presence of MRs in the aorta and the vena cava by
aldosterone binding assays. As a reference, the binding
of tritiated aldosterone to the cytosol of kidney and
salivary glands was also examined in the same ailimal.
The binding of [3H]aldosterone to MRs was measured
by means of glycerol density gradient analyses, and the
specificity was confirmed by competition with either
RU486 or HIOE, as described above. Under these
conditions, all cytosolic fractions were found to contain
MR that sedimented in the 8S-9S region (data not
0,8 -
Kd = 0.25 0.02 nM
Nmax = 22.1 0.9 fmol/mg protein
a
0,6 \- = 0.0089 0.0008
0,4 -
m ALDO
0,2
shown). The quantification of MR content in these
various tissues is given in Figure 8 The concentration of
aortic MR was slightly lower than the one estimated in
the heart. In accordance with immunohistochemical
data, specific aldosterone binding in the cytosol of the
vena cava was almost undetectable.
Discussion
Our study provides evidence for the presence of MRs
in the rabbit heart and large blood vessels. These
findings are supported by the results obtained by two
complementary methodological approaches. Biochemi-
cal methods have been used to characterize binding and
structural properties of the cardiovascular MR. Immu-
nohistochemistry has been performed to determine the
100-
RU 486
80 -
60 -
ZK91 587
40 -
20 -

o _ 1

0,0 0,5 1,0
ALDOSTERONE BINDING (nM)
FIGURE 5. Scatchard plot of aldosterone binding to rabbit
heart cytosol. Nmax, maximum number of binding sites. Cyto-
solic aliquots were incubated with increasing concentrations
of [J?Hlaldosterone (0.1-100 nM) for 4 hours at 4C. Bound
(B) and unbound (U) hormone were separated by dextran-
coated charcoal treatment. All points are means of triplicate
determinations.
-10 -9 - 8 -7 -h
LOG COMPETITOR CONCENTRATION (M)
FIGURE 6. Graph showing specificity of tritiated aldoste-
rone binding in rabbit heart cytosol. Cytosol was incubated for
4 hours at 4C with I nM [3Hlaldosterone either alone or in
the presence of increasing concentrations of unlabeled com-
petitors: aldosterone (ALDO, 0), spironolactone (SC9420, n),
ZK91587 (A), and RU486 (o). Results are expressed as the
percentage of binding measured with tritiated aldosterone
alone (16.3 fmol/mg protein).
 508 Circulation Research Vol 71, No 3 September 1992

A OSA M A BSA M arteries4,7; and in human vascular smooth muscle cells in

4 4 culture.6 In the present study, we have shown that
cardiovascular MRs possess characteristics virtually
identical to those of the rabbit kidney.29 In the cytosolic
fraction of the rabbit heart and aorta, we found a
o; 4- specific binding of aldosterone to one class of high
z affinity binding sites whose mineralocorticoid specificity
3- was clearly demonstrated by competition experiments.
0
The amount of MR present in the heart is relatively
't2- high, only two times lower than in the kidney, which is
a
the main site of action of aldosterone. In addition,
ultracentrifugation analysis together with immunologi-
cal probes identified the cardiac MR as a hetero-
oligomeric entity that includes hsp90, which is consid-
10

FRACTION NUMBER
0 10
FRACTION NUMBER
FIGURE 7. Graphs showing sedimentation gradient analyses
of aldosterone-rabbit mineralocorticoid receptor complexes.
Panel A: Cytosol prepared in TEGW buffer was incubated
with 5 nM [3Hlaldosterone alone (a) or in the presence of the
Downloaded from http://circres.ahajournals.org/ by guest on October 11, 2017
20
ered to play an important role in the activation process
of MRs.2430 The structural similarity between cardiac
MRs and MRs from other classic aldosterone target
tissues, such as the kidney or the colon, suggests that in
the cardiovascular system MRs might function through
the same general transduction pathway.

monoclonal anti-idiotypic antibody HJOE (diluted ascites at a
ratio of 1/200) (o). After charcoal dextran treatment, ali-
quots were layered onto the top of a 15-40% glycerol gradient
prepared in TEW buffer. Panel B: After charcoal treatment,
aldosterone-labeled cytosolic fractions were incubated for an
additional 4 hours at 4C with either a mouse immunoglobulin
G] control (c) or the monoclonal anti-heat shock protein 90
antibody 7C10 (diluted ascites at a ratio of1/50) ([) and layered
onto the top of glycerol gradients. Gradients were centrifuged
at 4C for 18 hours at 50,000 rpm. External standards were
myoglobin (M, 2S region), bovine serum albumin (BSA, 4.6S
region), and aldolase (A, 7.9S region).
cellular distribution of MRs within the cardiovascular
system. Aldosterone binding has been identified previ-
ously in the rat heart2,3; in rabbit,5 bovine,8 and rat
40
30
c
-0
D -6
20
0
The present immunohistochemical study allows us to
describe the tissue and cellular distribution of MRs. By
using the monoclonal antibody H1OE,19 a positive im-
munostaining was detected in the atria, ventricles,
aorta, and pulmonary artery. The antibody H1OE is an
anti-idiotypic antibody that possesses internal image
properties of aldosterone; i.e., its binding to the MR
could be easily inhibited by an excess of MR ligands.
This property is unique in that one can clearly attribute
immunoreactivity to MRs by in situ competition exper-
iments. Extinction of the immunostaining by preincuba-
tion with mineralocorticoid ligands, such as aldosterone,
associated with the absence of extinction by synthetic
ligands with no affinity for MRs, such as RU486, allows
us to assess mineralocorticoid specificity of the immu-
nostaining. Beyond the direct demonstration of the
presence of MRs in the heart and large vessels, immu-
nohistochemistry brings important information, since it
identifies the cell type possessing the MR. It appears
clearly that the MR is not restricted to smooth muscle
cells and myocytes. It is also present in endothelial cells
of the heart and vessels and possibly in the fibrocytes.
Quantitative interpretation of immunohistochemical
experiments has to be considered with caution. How-
ever, comparison of the intensity of staining within cell

CD
E_
1
populations of the same slide, which reduces the vari-
E
x,,o- 11
11
ability originating from different experimental pro-
I 10 11
11
cesses, allows valuable observations. It appears that the
111 range of intensity of staining was as follows: atrium>

JLa ventricle > aorta> pulmonary artery. This apparent re-

oo
111

O 1 , v 'Ei a * 1.'w .: * v IIE .5rO

K SG H A
FIGURE 8. Bar graph showing quantification of min-
eralocorticoid receptor in various rabbit tissues. Cytosolic
fractions of adrenalectomized rabbit kidney (K), salivary
glands (SG), heart (H), aorta (A), and vena cava (VC) were
incubated for 4 hours at 4C with 5 nM [3H]aldosterone alone
(open bar), in the presence of a 100-fold excess of RU486
(crosshatched bar), or in the presence of a 100-fold excess of
RU486 and the monoclonal anti-idiotypic antibody HJOE
(diluted ascites at a ratio of 1/200) (horizontally striped bar).
Quantification of steroid binding was achieved by measuring
the radioactivity recovered in the 8S-9S region in the sedi-
mentation profile of gradient analysis.
gional variation could actually reflect differences in the
VC abundance of MRs among these tissues. Such an inter-
pretation is in good agreement with our observation of
a specific aldosterone binding higher in the heart than in
the aorta.
When immunoreactivity in vessels of different size is
compared, it appears that the intensity of staining
decreases with the size of the arteries. The immuno-
staining is higher in the aorta and pulmonary artery
than in the carotid, renal, and mesenteric arteries and is
virtually undetectable in small arterioles and capillaries.
The absence of staining probably reflects an actual
absence or very low level of MRs in small intramyocar-
dial and intrarenal arterioles, for which direct compar-
isons are possible with the strong staining of the sur-
 Lombe's et al Cardiovascular Mineralocorticoid Receptor
rounding tissues (from the same slide) such as myocytes
and the renal cortical collecting duct, respectively. By
contrast, for the reasons explained above, the fact that
different histological sections were used to examine
large and medium-sized arteries precludes a firm con-
clusion concerning the relative abundance of MRs in
these vessels.
We could not detect any significant difference in the
immunostaining patterns between normal and adrenal-
ectomized rabbits. Nevertheless, limitation in quantita-
tive interpretation of immunohistochemical studies
does not rule out the possibility of a hormonal regula-
tion of cardiovascular MR expression. However, it is
noticeable that such an absence of steroid effects on
renal MR expression has already been reported with
immunohistochemical techniques by us20 and others.31
In contrast with corroborating evidence of the pres-
ence of MRs in the cardiovascular system, the nature of
specific aldosterone actions in the heart and blood
vessels is still under debate. Studies on direct effects of
Downloaded from http://circres.ahajournals.org/ by guest on October 11, 2017
mineralocorticoids on cardiac activity remain rather
limited. In the heart, aldosterone has been proposed to
exert direct inotropic effects,18,32 but the mechanisms of
these actions remain to be identified. In addition, in vivo
studies have reported that aldosterone might influence
myocardial structure by participating in the develop-
ment of cardiac fibrosis during ventricular hypertro-
phy.17 Along this line, our observation of MR-contain-
ing cells in the subendothelial connective layer of the
heart might be of interest and requires further studies.
Recently, aldosterone has been shown to modulate gene
expression of the Na+,K+-ATPase al-subunit and intra-
cellular electrolyte content in rat cardiocytes.16'33 These
results, together with the present demonstration of MRs
in the heart, support the proposal of direct actions of
aldosterone on heart functions.
In blood vessels, several studies have reported an
influence of aldosterone on sodium movements across
the cell plasma membrane. However, at the present
time, no general agreement has been reached concern-
ing the nature and the mechanism of these mineralocor-
ticoid actions. Both membrane permeability to sodium
and active NaX,K+-ATPase-dependent cell sodium ex-
trusion have been proposed to participate in the aldo-
sterone-dependent cellular ion modifications. Free in-
tracellular sodium content in smooth muscle cells has
been shown to be decreased,1213 unchanged," or in-
creased34 by aldosterone. Whereas aldosterone seems to
increase membrane permeability to sodium, it results in
either a rise in sodium influx into the cellular compart-
ment1"'34 or a rise in sodium efflux from the cell to the
extracellular compartment.14"5 Most of these studies
reported that aldosterone increases the active Na+,K+-
ATPase-dependent sodium extrusion, except one.35
Thus, although all these studies point out an important
impact of aldosterone on sodium movements in arterial
walls, the nature of these effects is still debatable.
Several factors may contribute to the variability of the
results, such as differences in the species studied, the
experimental approach, and the nature and the dose of
the mineralocorticoid hormone used. Moreover, a sup-
plementary degree of complexity has to be taken into
consideration for the interpretation of these physiologi-
cal studies. Indeed, interactions of sodium movements
with other ions, such as calcium or potassium,1""15'34 as
509
well as interactions of aldosterone with other hormonal
systems, such as vasopressin, angiotensin, and catechol-
amines 9,10,36-38 have been repeatedly reported in vessels.
On the whole, whereas one can expect an effect of
mineralocorticoid hormones on sodium movements in
cardiovascular cells possessing the MR, further studies
are required to determine its precise nature and
mechanism.
Recently, it has been shown that the expression of
selective mineralocorticoid action in MR-containing
cells depends on the presence of an enzyme (ll1-
hydroxysteroid dehydrogenase) degrading natural glu-
cocorticoids.39'40 Consequently, aldosterone effects can
be clearly evidenced in tissues possessing this enzyme,
e.g., the renal tubule,41 colon,42 and toad bladder.43 By
contrast, the presence of 11-hydroxysteroid dehydro-
genase activity in the heart is still controversial: some
authors did not detect it40,44 and others reported it.45-48
Concerning the vascular system, the presence of 11/3-
hydroxysteroid dehydrogenase activity was reported in
the rat aorta, caudal artery,48 and the mesenteric ar-
tery.7'48 This enzyme activity that permits aldosterone
access to MRs varies in relation to the availability of the
cofactor NADP in the preparation48 and also varies
from one cardiovascular tissue to another.48 Such vari-
ations could be at the origin of some discrepancies
concerning the regulatory effect of aldosterone on ion
movements.
Finally, the possibility exists that some aldosterone
actions might be mediated independent of the classical
receptor pathway. This could be suggested for the heart
and vessels as it has already been reported for other
steroid hormones, in particular, the central nervous
system.49 Along this line, the finding that aldosterone
very rapidly stimulates the sodium-proton exchanger50
might be of critical importance in determining the
action of aldosterone on the cardiovascular system.
In conclusion, we have demonstrated the presence of
MRs in the rabbit heart and vessels. Immunohistochem-
ical methods localize this receptor in the endothelial
cells, myocytes, and vascular smooth muscle cells of the
arterial walls. The precise actions and mechanisms of
action of aldosterone in these cells remain to be clarified.
Acknowledgments
We are indebted to Francine Delahaye for her skilled
technical assistance and to Dr. J. Perennec for helping us in
the interpretation of the histological data.
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 Immunohistochemical and biochemical evidence for a cardiovascular mineralocorticoid
receptor.
M Lombs, M E Oblin, J M Gasc, E E Baulieu, N Farman and J P Bonvalet
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Circ Res. 1992;71:503-510

doi: 10.1161/01.RES.71.3.503
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