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Endocrinology 147(5):2526 –2534
Copyright © 2006 by The Endocrine Society
doi: 10.1210/en.2005-1167
Spironolactone Preserves Cardiac Norepinephrine
Reuptake in Salt-Sensitive Dahl Rats
Sebastian J. Buss,* Johannes Backs,* Michael M. Kreusser, Stefan E. Hardt, Christiane Maser-Gluth,
Hugo A. Katus, and Markus Haass
Departments of Cardiology (S.J.B., J.B., M.M.K., S.E.H., H.A.K.) and Pharmacology (C.M.-G.), University of Heidelberg,
69120 Heidelberg, Germany; and Department of Cardiology, Theresienkrankenhaus (M.H.), 68165 Mannheim, Germany
An impairment of cardiac norepinephrine (NE) reuptake via
the neuronal NE transporter (NET) enhances the effects of
increased cardiac NE release in heart failure patients. Increasing evidence suggests that aldosterone and endothelins
promote sympathetic overstimulation of failing hearts. Saltsensitive Dahl rats (DS) fed a high-salt diet developed arterial
hypertension and diastolic heart failure as well as elevated
plasma levels of endothelin-1 and NE. Cardiac NE reuptake
and NET-binding sites, as assessed by clearance of bolusinjected [3H]NE in isolated perfused rat hearts and [3H]mazindol binding, were reduced. Treatment of DS with the mineralocorticoid receptor antagonist spironolactone preserved
the plasma levels of endothelin-1 and NE, cardiac NE reuptake, and myocardial NET density. Moreover, the ventricular function and survival of spironolactone-treated DS were
significantly improved compared with untreated DS. The ␣1-
T
HE RENIN-ANGIOTENSIN-aldosterone system plays a
central role in the pathophysiology of heart failure
(1–5). Its activation is associated with a poor prognosis of
heart failure patients (6, 7). Since the Randomized Aldactone
Evaluation Study (3) revealed a reduced mortality of heart
failure patients treated with the mineralocorticoid receptor
(MR) antagonist spironolactone, it has become of interest to
determine how MR antagonism improves survival. Aldosterone, the endogenous MR agonist, is known to not only
regulate renal electrolyte and body fluid balance, but also
promote cardiac and vascular fibrosis, baroreceptor dysfunction, parasympathetic inhibition, and sympathetic activation
(5, 8). However, it remained unclear which action of MR
antagonism contributes in particular to an improved survival
rate of heart failure patients. Because in clinical studies the
etiology of heart failure is heterogeneous, as are the type and
dosages of background medications, it is difficult to conclude
from clinical data the underlying mechanisms. Therefore,
First Published Online January 26, 2006
* S.J.B. and J.B. contributed equally to this work and should both be
considered as first authors.
Abbreviations: (⫹)dP/dtmax, Left ventricular contractility; (⫺)dP/
dtmax, left ventricular relaxation; DS, Dahl rat; ET, endothelin; ETA, ET-A
receptor; LVEDP, left ventricular end-diastolic pressure; MIBG, metaiodobenzylguanidine; MR, mineralocorticoid receptor; NE, norepinephrine; NET, norepinephrine transporter; proANP, prohormone form of
atrial natriuretic peptide.
Endocrinology is published monthly by The Endocrine Society (http://
www.endo-society.org), the foremost professional society serving the
endocrine community.
inhibitor prazosin decreased blood pressure in DS similar to
spironolactone treatment, but did not normalize the plasma
levels of endothelin-1 and NE, NE reuptake, or ventricular
function. In a heart failure-independent model, Wistar rats
that were infused with aldosterone and fed a high-salt diet
developed impaired cardiac NE reuptake. Treatment of these
rats with the endothelin A receptor antagonist darusentan
attenuated the impairment of NE reuptake. In conclusion,
spironolactone preserves NET-dependent cardiac NE reuptake in salt-dependent heart failure. Evidence is provided
that aldosterone inhibits NET function through an interaction with the endothelin system. Selective antagonism of the
mineralocorticoid and/or the endothelin A receptor might
represent therapeutic principles to prevent cardiac sympathetic overactivity in salt-dependent heart failure. (Endocrinology 147: 2526 –2534, 2006)
experimental heart failure models with a homogenous etiology and pathophysiology are helpful tools to dissect aldosterone actions leading to morbidity and mortality. Saltsensitive Dahl rats (DS) are an established model for heart
failure with predominant diastolic dysfunction and have
been shown to develop an activation of the local cardiac
aldosterone system (9 –13).
Activation of the sympathetic nervous system is a major
characteristic in heart failure patients (14). Elevated plasma
levels of norepinephrine (NE) are associated with a poor
prognosis of heart failure patients (15). An impairment of
cardiac NE reuptake by NET down-regulation contributes to
an increased cardiac net release of NE in heart failure, which
is associated with a depletion of cardiac NE stores, downregulation of cardiac ␤-adrenoceptors, and profound alterations of postreceptor signal coupling (16 –21). Clinical studies have shown that impaired cardiac NE reuptake is also
associated with a poor prognosis of heart failure patients
(reflected by worsening of heart failure and increasing incidence of sudden death) (22, 23). Recently, we have shown
that endothelin (ET)-1 is a potent negative regulator of NET
and that inhibition of the ET-A receptor (ETA) attenuates
NET impairment in aortic-banded rats (24). Interestingly,
increasing evidence suggests an interaction between the aldosterone and ET systems (25).
In the present study we tested the hypothesis that MR
antagonism improves cardiac sympathetic nerve function in
DS (11). Determinants of cardiac NE homeostasis and left
ventricular function were evaluated. Moreover, we asked
whether aldosterone mediates its effects on cardiac sympa-
2526
Buss et al. • Spironolactone and NE Reuptake
thetic nerve function through an interaction with the endothelin system.
Materials and Methods
This investigation conformed with the Guide for the Care and Use of
Laboratory Animals published by the National Institutes of Health and
was approved by the authorities of the Regierungspräsidium Karlsruhe,
Germany.
Dahl salt-resistant (DR) and -sensitive (DS) rats
Male DR and DS (9 wk of age; mean weight, 200 g; M&B, Ry, Denmark) were fed a high-salt diet (8% NaCl) for 40 d. DS were randomized
to receive either spironolactone (50 mg/kg䡠d; DS ⫹ spironolactone) or
prazosin (5 mg/kg䡠d; DS ⫹ prazosin) in the drinking water (was
changed twice the day) beginning with onset of the diet. Water and drug
intake was controlled daily.
Aldosterone infusion
Male Wistar rats (Charles River Laboratories, Sulzfeld, Germany;
mean weight, 200 g) were fed on a high-salt diet (8% NaCl) and simultaneously received 0.75 ␮g/h d-aldosterone (Fischer Scientific Germany,
Schwerte, Germany) via implanted osmotic minipumps (model 2004,
Alzet Co., Charles River Laboratories, Sulzfeld, Germany) for 28 d as
previously described (25). One group (aldosterone ⫹ darusentan) was
treated with the specific ETA antagonist darusentan (Abbott Laboratories, Ludwigshafen, Germany; 30 mg/kg䡠d) in the drinking water. The
control group received vehicle and standard chow (0.2% NaCl).
Hemodynamic studies
Rats were anesthetized with pentobarbital (50 mg/kg, ip), and a
polyethylene catheter (Aorta 24; Medex Medical, Klein-Winternheim,
Germany) was implanted into the abdominal aorta, sc tunneled to the
suprascapular area, and brought out through a steel tether that allowed
the animals free movement and access to water during the recovery
period and the experiments. Using this catheter, arterial blood pressure
was recorded with a transducer (Statham P 23 XL; Spectramed, Oxnard,
CA) connected to a PowerLab personal computer system (ADInstruments Pty. Ltd., Colorado Springs, CO). Blood pressure was recorded
after a 45-min rest of the awake animal in a dark, quiet room, and mean
arterial blood pressure and heart rate (beats per minute) were calculated.
Intraventricular pressure was recorded as previously described (26). The
left ventricular end-diastolic pressure (LVEDP), left ventricular contractility [(⫹)dP/dtmax] and left ventricular relaxation [(⫺)dP/dtmax] were
calculated using PowerLab Chart software (ADInstruments Pty. Ltd.).
Endocrinology, May 2006, 147(5):2526 –2534
2527
12; mean ⫾ sd). The intraassay coefficient of variation was 3.5– 8.5%, and
the interassay coefficient of variation was 9.6 –12.2%.
NE tissue levels
The left ventricle was rinsed in ice-cold 0.9% saline and frozen in
liquid nitrogen until HPLC and electrochemical detection as previously
described in detail (30, 31).
Isolated heart perfusion
Wistar rats were anesthetized with thiopental (100 mg/kg, ip). The
hearts were rapidly cut out and rinsed in ice-cold buffer, and the aorta
was cannulated for perfusion according to the method described by
Langendorff (32). Within one experiment, eight to 12 spontaneously
beating hearts were perfused simultaneously at a constant coronary flow
and a constant temperature of 37.5 C. The perfusion medium was a
modified Krebs-Henseleit solution (125 mmol/liter NaCl, 16.9 mmol/
liter NaHCO3, 0.2 mmol/liter Na2HPO4, 4.0 mmol/liter KCl, 1.85
mmol/liter CaCl2, 1.0 mmol/liter MgCl2, 11 mmol/liter glucose, and
0.027 mmol/liter EDTA). The buffer was gassed with 95% O2 and 5%
CO2, and the pH was adjusted to 7.4. Cardiac [3H]NE uptake was
determined as described previously (16). Briefly, a bolus of [3H]NE (1 ml,
3 ␮Ci, 100 pmol NE; Amersham-Buchler, Braunschweig, Germany) was
injected into the perfusion system and proportionally distributed to the
hearts and blank channels. Radioactivity was measured in the effluent.
The amount of [3H]NE extracted by the hearts (uptake) was expressed
as the percentage of radioactivity measured in the blank channels.
[3H]Mazindol binding
Plasma membranes of right and left ventricles were prepared as
described previously (16). Radioligand binding assays were performed
in a total volume of 250 ␮l containing 50 ␮g plasma membranes and
increasing concentrations of [3H]mazindol (specific activity, 52,5 Ci/
mmol; NEN Life Science Products, Dreieich, Germany) as a specific
ligand. Nonspecific binding was determined by measuring the residual
binding in the presence of desipramine (100 ␮mol/liter). The incubation
was carried out at 30 C and was terminated by rapid vacuum filtration
through a MultiScreenHTS-FB filter plate (Millipore Corp., Schwalbach,
Germany). All experiments were performed in triplicate. The remaining
filter radioactivity was determined, and the binding capacity was calculated using PRISM version 4.00 software (GraphPad, Inc., San Diego,
CA).
Echocardiography
Transthoracic echocardiography was performed as previously described in detail (33). The investigator who conducted the echocardiography was blinded to the treatment status.
Determination of plasma neurohormones
Survival analysis
For measurement of plasma NE, the animals were put into individual
cages in a quiet room between 1700 –1900 h, and the implanted catheter
was connected to a syringe. Forty-five minutes later, a 400-␮l blood
sample was taken from the awake animal and was replaced by an equal
volume of saline. The plasma concentration of NE was determined by
a radioenzymatic assay as previously described (27). Plasma concentrations of ET-1, the stable N-terminal part (amino acids 1–98) of the
prohormone form of atrial natriuretic peptide (proANP), and aldosterone were determined in venous blood samples taken from the femoral
vein. ET-1 and proANP were determined using enzyme immunoassays
according to the manufacturer’s instructions (Biomedica, Vienna, Austria). Aldosterone was measured using a specific in-house RIA established at the Steroid Laboratory of University of Heidelberg, using
tritiated aldosterone ([1,2,4,6-3H]aldosterone; Amersham Biosciences,
Freiburg, Germany) and an antibody raised and characterized in the
steroid laboratory as described previously (28, 29). Before RIA a recovery-corrected extraction and chromatographic purification were performed, thereby efficiently removing cross-reaction steroids. The standard curve ranged from 1–200 pg/tube, and the sensitivity was 2 pg/
tube (1 ng/100 ml). The recovery of a known amount of aldosterone
determined repeatedly in quality control samples was 103.8 ⫾ 8.2% (n ⫽
DS were treated as described above (DS, DS ⫹ spironolactone, or
DS ⫹ prazosin). Ten rats per group were monitored, and deaths were
recorded every day. Survival was described by standard Kaplan-Meier
analysis.
Statistics
The results are expressed as the mean ⫾ sem. Statistical analysis was
performed using SPSS 12.0 software (SPSS, Inc., Chicago, IL). Differences
between groups were tested by one-way ANOVA with post hoc comparisons by Fisher’s protected least significant difference test or unpaired Student’s t test where appropriate. Kaplan-Meier survival analysis was performed using the log-rank test. In all tests, P ⬍ 0.05 was
considered statistically significant.
Results
Effects of spironolactone on heart failure indices in DS
After 40 d of a high-salt diet, DS showed severe arterial
hypertension, with a doubled mean arterial blood pressure
2528
Endocrinology, May 2006, 147(5):2526 –2534
Buss et al. • Spironolactone and NE Reuptake
FIG. 1. Hemodynamic
characterization of DR and DS after 40 d of a 8%
NaCl diet. Beginning with the onset of
the high-salt diet, some DS rats were
treated with either spironolactone (SP)
or prazosine (P) as a blood pressure control. Mean arterial blood pressure
(MAP; A), LVEDP (B), (⫹)dP/dtmax (C),
and (⫺)dP/dtmax (D) are shown. n.s., Not
significant.
compared with DR (Fig. 1A). Moreover, DS developed signs
of heart failure, as indicated by biventricular myocardial
hypertrophy combined with elevated lung wet weights (Table 1) and elevated LVEDPs (Fig. 1B). Treatment of these
animals with spironolactone lowered the mean arterial blood
pressure by only 15%, but markedly attenuated biventricular
hypertrophy and pulmonary congestion (Table 1). In contrast, treatment with prazosin, which causes a similar effect
on arterial blood pressure as spironolactone (Fig. 1A), did not
result in a comparable attenuation of organ weights/body
weight ratios (Table 1). By inserting a cannula connected to
a pressure transducer via a short (⬍10 cm) water-filled polyethylene catheter, we measured the left ventricular pressure
of these animals. The use of this method to assess LVEDP and
especially (⫹)dP/dtmax has a limitation in that the distance
between the LV and the pressure transducer may result in
false low values. However, this method is suitable to compare the effects of different drugs, because this potential error
would be a systemic error, affecting all groups equally. Treatment of DS with spironolactone, but not with prazosine,
prevented the increase in LVEDP (Fig. 1B) but attenuated
(⫹)dP/dtmax only partially (Fig. 1C). In accordance with
previous findings by others (10, 11) that salt-induced heart
failure of DS rats depends on a marked diastolic dysfunction,
we observed a markedly impaired (-)dP/dtmax (Fig 1D).
Treatment with spironolactone, but not with prazosine, pre-
TABLE 1. Basic characterization of DR and DS after 40 d on an 8% NaCl diet
BW (g)
Heart weight/BW (mg/g)
LV/BW (mg/g)
RV/BW (mg/g)
Lung wet weight/BW (mg/g)
Heart rate (beats/min)
DR
DS
DS ⫹ SP
DS ⫹ P
312 ⫾ 7
(25)
4.07 ⫾ 0.07
(25)
3.19 ⫾ 0.04
(10)
0.60 ⫾ 0.02
(10)
5.85 ⫾ 0.12
(21)
400 ⫾ 10
(8)
252 ⫾ 5a
(41)
8.06 ⫾ 0.23a
(40)
6.09 ⫾ 0.17a
(28)
1.05 ⫾ 0.03a
(28)
13.02 ⫾ 0.88a
(40)
427 ⫾ 10
(7)
341 ⫾ 10a,b
(23)
5.58 ⫾ 0.21a,b
(23)
4.35 ⫾ 0.17a,b
(23)
0.67 ⫾ 0.03b
(23)
7.01 ⫾ 0.65b
(23)
439 ⫾ 19
(8)
275 ⫾ 10a,b,c
(9)
7.06 ⫾ 0.29a,b,c
(9)
5.90 ⫾ 0.28a,c
(9)
0.96 ⫾ 0.07a,c
(9)
9.52 ⫾ 1.78a,b,c
(9)
439 ⫾ 8a
(7)
Beginning with the onset of the high-salt diet, some DS rats were treated with either spironolactone (SP) or prazosine (P) as a blood pressure
control. BW, Body weight; LV, left ventricle; RV, right ventricle. The number of rats is in parentheses.
a
P ⬍ 0.05 vs. DR.
b
P ⬍ 0.05 vs. DS.
c
P ⬍ 0.05 vs. DS ⫹ SP.
Buss et al. • Spironolactone and NE Reuptake
Endocrinology, May 2006, 147(5):2526 –2534
2529
vented an impaired (-)dP/dtmax in DS (Fig. 1D). Likewise,
transthoracic echocardiography revealed that treatment with
spironolactone prevented an increase in anterior wall thickness in DS, but only affected marginally the decrease in
ventricular fractional shortening (Table 2).
Effects of spironolactone on survival of DS
To determine whether spironolactone-mediated beneficial
effects on cardiac function also lead to an improved outcome
for DS, a Kaplan-Meier survival (10 animals/group) analysis
was performed. Treatment with spironolactone prolonged
the median survival of DS to 72 d after onset of the diet
compared with 42 d in nontreated and prazosin-treated DS
(Fig. 2). However, spironolactone treatment of DS did not
entirely prevent, but delayed, mortality of DS in response to
a high-salt diet. No spironolactone-treated DS rats lived
longer than 102 d after onset of the diet. The postmortem
analysis of both spironolactone- and non/prazosine-treated
animals revealed signs of heart failure (ascites, pleural effusion, and enlargement of LV and RV).
Effects of spironolactone on neurohormonal indices in DS
Elevated NE plasma levels in DS compared with DR indicated activation of the sympathetic nervous system (Fig.
3A). We observed higher plasma levels of ET-1 in DS compared with DR, indicating activation of the endogenous ET
system (Fig. 3B). In accordance, a similar increase in plasma
ET-1 was reported by others (10, 11). Although plasma levels
of aldosterone were comparable in DR and DS that were fed
a standard diet (⬃13 ng/dl in both groups; data not shown),
high salt intake induced a reduction of plasma aldosterone
in DR (⬍5 ng/dl), but not in DS (11 ng/dl; Fig. 3C), suggesting that the endogenous aldosterone system of DS is
unresponsive to salt. Consistently, Nishikimi et al. (9) reported doubled plasma aldosterone levels in DS compared
with DR. As a marker for the hemodynamic overload of the
heart, proANP plasma levels were 10-fold increased. Treatment with spironolactone, but not prazosine, prevented increased plasma levels of both NE and ET-1 (Fig. 3, A and B).
ProANP plasma levels were attenuated by spironolactone
and, although less pronounced, by prazosine (Fig. 3D). As
expected, treatment with spironolactone elevated plasma aldosterone levels in DS, indicating efficient MR antagonism
by spironolactone (Fig. 3C). In accordance, a similar increase
in plasma aldosterone due to spironolactone was observed
by others (34).
TABLE 2. Echocardiography of DR (n ⫽ 5) and DS (n ⫽ 5) after
40 d on a 8% NaCl diet
DR
DS
DS ⫹ SP
Heart rate (beats/min)
323 ⫾ 15 322 ⫾ 33
381 ⫾ 24
Fractional shortening (%)
40.8 ⫾ 1.7 33.3 ⫾ 1.3a 34.8 ⫾ 1.8
End-diastolic diameter (mm) 7.8 ⫾ 0.3 7.5 ⫾ 0.4
7.6 ⫾ 0.4
End-systolic diameter (mm)
4.6 ⫾ 0.3 5.0 ⫾ 0.3
5.0 ⫾ 0.3
Diastolic anterior wall
1.8 ⫾ 0.1 2.9 ⫾ 0.2a 1.8 ⫾ 0.07b
thickness (mm)
Beginning with the onset of the high-salt diet, some DS rats were
treated with spironolactone (DS ⫹ SP; n ⫽ 5).
a
P ⬍ 0.05 vs. DR.
b
P ⬍ 0.05 vs. DS.
FIG. 2. Kaplan-Meier survival curves of DS (n ⫽ 10), spironolactonetreated DS (DS ⫹ SP; n ⫽ 10), and prazosin-treated DS (DS ⫹ P; n ⫽
10) during a 8% NaCl diet. The arrow above the diagram indicates the
duration of the 8% NaCl diet. *, P ⬍ 0.05 vs. DS and DS ⫹ P.
Effects of spironolactone on cardiac NE homeostasis in DS
To test the hypothesis that an endogenously activated
aldosterone system in DS impairs NE reuptake in vivo, the
uptake of exogenous [3H]NE in a Langendorff preparation
was determined. Cardiac [3H]NE uptake was reduced by
approximately 30% in DS compared with DR, and in fact,
treatment with spironolactone, but not prazosin, preserved
cardiac NE reuptake in DS (Fig. 4). There was no difference
between DR, DS, DS ⫹ spironolactone, and DS ⫹ prazosine
rats in the residual uptake of [3H]NE after specific blockade
of NET with desipramine, indicating that the diminished
cardiac elimination of [3H]NE was entirely due to reduced
uptake via the NET and not, for example, to the extraneuronal NE transporter (uptake2 carrier; Fig. 4). Impaired cardiac NE reuptake might induce depletion of cardiac NE
stores (16), reflecting an overactivity of the cardiac sympathetic nervous system. Tissue NE stores in the right and left
ventricles (Fig. 5, A and B) were markedly depleted in DS
compared with DR. Treatment with spironolactone, but not
prazosin, attenuated NE depletion in the left ventricle only
partially, but prevented it in the right ventricle (Fig. 5, A and
B). This discrepancy led us to ask whether aldosterone impairs NE reuptake only in the right ventricle. We reported
previously that in aortic banded rats, impaired NE reuptake
of the whole isolated perfused heart is caused by reduced
NET density in the right and left ventricles (16, 24). Consistently, NET density, as assessed by [3H]mazindol binding,
was also reduced in both ventricles of DS compared with DR
rats. However, in contrast to the NE stores, spironolactone
normalized NET density in the right and left ventricles (Fig.
5, C and D).
Effects of ETA antagonism on NE reuptake in aldosteroneinfused Wistar rats
To determine whether impaired NE reuptake is directly
associated with the aldosterone system, we used an experimental animal model that mimics activation of the aldosterone system. Male Wistar rats received aldosterone via osmotic minipumps and simultaneously a high-salt diet for
28 d. Because these animals did not develop heart failure, it
is unlikely that potential effects on NE reuptake were me-
2530
Endocrinology, May 2006, 147(5):2526 –2534
Buss et al. • Spironolactone and NE Reuptake
FIG. 3. Plasma levels of NE (A), ET-1
(B), aldosterone (AL; C), and proANP
(D) in DR, DS, DS ⫹ SP, and DS ⫹ P rats
after 40 d of an 8% NaCl diet. n.s., Not
significant.
diated by activation of other heart failure-associated neurohormonal systems. Sufficient application of aldosterone via
osmotic minipumps was confirmed by the almost tripled
plasma aldosterone levels compared with control rats (vehicle infusion and normal salt diet; Fig. 6A). Consistently,
comparable levels were reported by others (34). Compared
with control animals, in aldosterone-infused rats we observed impaired cardiac [3H]NE uptake (Fig. 5B). This result
confirmed that activation of the aldosterone system is sufficient to inhibit NET function. Treatment with the specific
ETA receptor antagonist darusentan did not attenuate increased plasma aldosterone levels (Fig. 6A), indicating that
in this model ETA antagonism does not prevent activation of
the aldosterone system, e.g. by inducing its degradation.
Despite high aldosterone levels, treatment of aldosteroneinfused rats with darusentan attenuated a decreased elimination of NE via NET (Fig. 6B), suggesting that activation of
ETA is required for aldosterone-mediated impairment of
NET.
Discussion
FIG. 4. Cardiac [3H]NE uptake with (⫹) and without (⫺) blockade of
NET with desipramine (DMI) in DR, DS, DS ⫹ SP, and DS ⫹ P rats
after 40 d of an 8% NaCl diet. n.s., Not significant.
We have previously shown that an impairment of NE
reuptake in experimental heart failure is mediated by posttranscriptional down-regulation of neuronal NET within the
failing heart (16). More recently, we provided evidence that
ETA activation via ET-1 negatively regulates NE reuptake,
and that activation of the endogenous ET system mediates
NET down-regulation in heart failure (24). However, other
groups demonstrated that aldosterone might also be involved in the regulation of NET function (1, 35). In the
present study we confirm that MR antagonism attenuates
cardiac sympathetic dysfunction and improves cardiac contractile function in an experimental model of salt-induced
heart failure. In addition, we show for the first time that
ETA activation contributes to aldosterone-induced NET
impairment.
Despite a similar effect on arterial blood pressure, only
treatment with spironolactone, but not the ␣1-inhibitor prazosin, significantly improves the cardiac function and sur-
Buss et al. • Spironolactone and NE Reuptake
Endocrinology, May 2006, 147(5):2526 –2534
2531
FIG. 5. Left (A) and right (B) ventricular tissue concentrations of NE in DR, DS, DS ⫹ SP, and DS ⫹ P rats after 40 d of an 8% NaCl diet. Cardiac
[3H]mazindol binding in left (C) and right (D) ventricles of DR, DS, DS ⫹ SP, and DS ⫹ P rats after 40 d of an 8% NaCl diet is shown. n.s.,
Not significant.
vival of DS. In addition, compared with DR, the arterial blood
pressure of spironolactone-treated DS was still markedly
enhanced by approximately 60 mm Hg. This indicates that
the beneficial effects of spironolactone are blood pressure
independent. These findings are consistent with previous
experimental (36, 37) and clinical (2, 3) studies and reveal that
the aldosterone system plays a key role in the progression of
heart failure.
Our results demonstrate that MR antagonism normalizes
plasma NE levels and attenuates depletion of cardiac NE
FIG. 6. Plasma aldosterone (AL) levels
(A) and cardiac [3H]NE uptake (B) in
rats after 28 d of infusion with aldosterone via osmotic minipumps (0.75 ␮g
aldosterone/h) and a simultaneous diet
of 8% NaCl (AL) compared with control
animals that received the vehicle and
standard chow (0.2% NaCl) for 28 d.
Beginning with the onset of aldosterone
infusion and high-salt diet, some rats
were treated with darusentan (DA).
n.s., Not significant.
stores in salt-dependent heart failure. Both could be explained by a preservation of NE reuptake via the neuronal
NET. In this study we show that spironolactone prevents an
impairment of NE reuptake and a reduction of NET density,
suggesting that aldosterone inhibits NET function by downregulation of NET per sympathetic nerve ending. One could
argue that the spironolactone-induced preservation of cardiac NE reuptake in salt-sensitive Dahl rats was secondary
due to an improvement of cardiac function as a consequence
of other beneficial effects of spironolactone (e.g. antifibrotic
2532
Endocrinology, May 2006, 147(5):2526 –2534
effect) (36). In this regard, the observation that activation of
the aldosterone system via aldosterone infusion and a simultaneous high-salt diet also caused NET impairment supported the hypothesis that aldosterone, and not other independently activated neurohormonal systems, inhibit the
NET.
In accordance, using iodine-123-metaiodobenzylguanidine (MIBG) uptake, clinical studies (1, 35) provided indirect
evidence that spironolactone treatment caused an improvement of NE reuptake. Iodine-123-MIBG is thought to use the
same transport mechanisms as NE (38). However, this
method can distinguish neither between neuronal or extraneuronal uptake nor between reduced NE uptake or enhanced NE release. Because the present study performed
[3H]NE uptake measurements in the absence and presence of
the specific NET antagonist desipramine, we provide direct
evidence that spironolactone preserves NE reuptake via
NET.
Our recent findings that ET-1 inhibits NET in aortic
banded rats (24) and DS (our unpublished observations)
together with the present observation that aldosterone does
the same demonstrate that the possibility exists that ET-1
mediates its effect via aldosterone or vice versa. Because ET-1
exerts its effect on NET function in a rapid manner (24),
whereas the MR-mediated effect occurs in a chronic manner
and is only observed in the presence of a high-salt diet, it
seems more likely that aldosterone affects NET function
through salt-dependent activation of the ET system. Interestingly, it was demonstrated that aldosterone infusion to
salt-loaded rats induced vascular expression of ET-1, and
that MR antagonism normalized vascular ET-1 levels that
were increased in liquorice-induced hypertension (25, 39).
Likewise, we observed that spironolactone treatment of DS
normalizes plasma ET-1 levels. We have previously shown
that endogenous ET-1 inhibits NET function by selective
activation of ETA in experimental heart failure (24). In this
study we demonstrate that ETA antagonism attenuates saltdependent and aldosterone-induced impairment of NET in
a heart failure-independent model. One could argue that the
impairment of NET function in this model is a consequence
of high blood pressure, and the improvement after ETA antagonism is the result of lowered blood pressure. However,
high blood pressure alone is unlikely to cause NET impairment independent of salt loading and aldosterone infusion,
because in a salt-independent model of arterial hypertension,
we did not observe any NET impairment (40). Moreover, our
observation that DS rats still have a high mean arterial blood
pressure, by far higher than those of aldosterone-infused and
salt-loaded rats (25), after treatment with spironolactone, but
show normalized NE reuptake, strongly suggests that high
blood pressure per se is not a negative stimulus for NET
function. In fact, NET function seems to depend, rather, on
the activation status of the aldosterone system. We postulate
that aldosterone inhibits NET function in a salt-dependent
manner via activation of the ET system. Our findings that
both MR and ETA antagonism preserve NET function in heart
failure models suggest that activated aldosterone and ET
systems enhance cardiac sympathetic activity by inhibiting
NET-mediated NE reuptake.
In the present study the effect of spironolactone on left
Buss et al. • Spironolactone and NE Reuptake
ventricular NET density was more dramatic than the effect
on left ventricular cardiac NE stores. A potential explanation
for this discrepancy is that other aldosterone-independent
presynaptic mechanisms also contribute to the depletion of
cardiac NE stores. Likewise, we and others (19, 40, 41) reported that enhanced exocytotic NE release (e.g. by activation
of angiotensin receptors or inhibition of ␣2-receptors) also
causes sympathetic overdrive resulting in depleted cardiac
NE stores. Another possibility is that MR antagonism by
itself exerts dual effects on NE reuptake and exocytotic NE
release. Interestingly, it has been reported that MR antagonism induced a decrease in cardiac NE stores in the viable
myocardium of myocardial infarcted rats (42, 43). Although
the significance and underlying mechanism of this finding
remain elusive, these reports suggest that aldosterone might
also inhibit exocytotic NE release. In this regard, the interaction between the aldosterone and ET systems is of great
interest, because it was demonstrated that ET-1 not only
inhibits cardiac NE reuptake via ETA, but also attenuates
cardiac exocytotic NE release via ETB (24). Therefore, MR
antagonism could exert opposite effects on cardiac NE stores
via reduced production of ET-1 and, consequently, attenuated activation of ETA and ETB, explaining the relatively mild
restoration of left ventricular NE in DS in response to spironolactone. The finding that right ventricular NE stores
were completely normalized could be explained by the possibility that the right ventricle benefits more than the left
ventricle from a reduced volume overload in response to the
slight diuretic effect of spironolactone. As a consequence, the
right ventricle might be more relieved from the neurohormonal stress that induces exocytotic NE release.
Clinical implications
Myocardial NE reuptake is known to be a strong prognostic marker for overall mortality in heart failure (44). During the preparation of this manuscript, it was demonstrated
that adenoviral gene transfer of NET into failing hearts of
rabbits is sufficient to improve cardiac function (45). The
present study identified aldosterone as a potent negative
regulator of NE reuptake. Consequently, the beneficial effects of spironolactone on the survival of heart failure patients might be at least in part mediated by this effect. Likewise, improved NE reuptake would decrease the NE
concentration in the synaptic cleft and, therefore, would explain the finding of the Randomized Aldactone Evaluation
Study that spironolactone reduces the rate of sudden cardiac
death by elevating the threshold for ventricular fibrillation
(3). Thus, imaging techniques such as iodine-123-MIBG scintigraphy, which allow an assessment of NE reuptake, could
identify a patient population that benefits in particular from
MR antagonism. Moreover, our finding that aldosterone inhibits NE reuptake via activation of the ET system implies
that such a patient population could also benefit from ETA
antagonism. The identification of heart failure subpopulations that benefit from ETA antagonism is of particular interest, because, to date, clinical trials using ET receptor antagonists could not demonstrate an improvement in
mortality in a broad heart failure population (46).
Buss et al. • Spironolactone and NE Reuptake
Endocrinology, May 2006, 147(5):2526 –2534
Conclusions
In conclusion, aldosterone inhibits NET-mediated cardiac
NE reuptake. This action of aldosterone is at least partially
mediated via an interaction with the ET system resulting in
ETA activation. In salt-dependent heart failure, MR antagonism normalizes plasma ET-1 levels and consequently preserves cardiac NE reuptake and plasma NE levels. These
findings provide insight into the regulatory mechanisms by
which aldosterone enhances cardiac sympathetic activity in
heart failure.
16.
17.
18.
19.
20.
Acknowledgments
The expert technical assistance of Silvia Harrack, Jutta Krebs, and
Michaela Oestringer is gratefully acknowledged. We thank Dr. Klaus
Muenter (Knoll AG, Ludwigshafen, Germany) for his generous gift of
darusentan.
21.
Received September 12, 2005. Accepted January 17, 2006.
Address all correspondence and requests for reprints to: Dr. Johannes
Backs, Department of Molecular Biology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas
75390-9148. E-mail: [email protected].
All authors have nothing to declare.
23.
22.
24.
25.
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