Full text of DOI:10.1007/BF03343899

J. Endocrinol. Invest. 24: 587-597, 2001
Hypoxia modulates rapid effects of aldosterone on
oxidative metabolism in human calf muscle
M. Christ*, J. Zange**, C.P. Janson*, K. Müller**, P. Kuklinski**, B.M.W. Schmidt*,
H.C. Tillmann*, R. Gerzer**, and M. Wehling*
*Institute of Clinical Pharmacology, Klinikum Mannheim, University of Heidelberg; **Institute of
Aerospace Medicine, DLR, Cologne, Germany
ABSTRACT. Non-genomic effects of aldosterone
on oxidative metabolism of skeletal muscle have
been shown, recently. To further characterize these
rapid effects on the increase of phosphocreatine
(PCr) in the recovery period after isometric exerci-
se, a randomized cross-over placebo-controlled
study was conducted on 9 healthy volunteers. Hy-
poxia was chosen to test the dependence of the
effect on oxygen supply and thus its relation to ox-
idative vs non-oxidative metabolism. Parameters
related to the energy metabolism of calf muscles
were measured by 31P magnetic resonance spec-
troscopy during four repetitive contractions in four
different tests (hypoxia [FiO2=0.13] vs normoxia
[FiO2=0.21], 0.5 mg aldosterone). The area-un-
der-curves of post-exercise PCr levels after the 4th
contraction were significantly increased by aldos-
terone vs placebo during normoxia (875.5 5.1 vs
857.2 8.3%·min; p=0.02). In addition, aldosterone
induced an undershoot of inorganic phosphate (Pi)
in the recovery after isometric exercise (77.5 5.4
vs 88.9 5.1 mmol/l·min; p=0.05). Hypoxia blocked
effects of aldosterone on PCr overshoot and Pi un-
dershoot. Concentrations of ATP, ADP, phospho-
monoesters, and intracellular pH were not affect-
ed by those interventions. In conclusion, these da-
ta demonstrate rapid actions of aldosterone on
post-excercise PCr and Pi levels in human calf mus-
cle, which are blocked by hypoxia. We hypothe-
size that aldosterone rapidly interferes with ox-
idative metabolism probably by direct modulation
of ATP-turnover, which is induced by an oxygen-
dependent imbalance of the ATP-synthesis and uti-
lization rate.
(J. Endocrinol. Invest. 24: 587-597, 2001)
©2001, Editrice Kurtis
INTRODUCTION
nomic actions of steroids on skeletal muscle (5) and
other targets (6) have been identified.
More than 3 decades ago the profound impact of
steroids on nuclear transcription and protein syn-
thesis was recognized, and molecular mechanisms
of genomic steroid effects in target tissues were
identified (1, 2). Steroid actions on skeletal muscle
and bone have been commonly explained by these
genomic mechanisms. For example, dexametha-
sone modulates the expression of GLUT1 and
GLUT4 protein in skeletal muscle cells due to de
novo synthesis (3), and glucocorticoid treatment
may induce mitochondrial dysfunction leading to
steroid myopathy (4). In addition, novel, non-ge-
Non-genomic effects of steroids are mainly char-
acterized by their rapid onset of action (<10 min).
Furthermore, inhibitors of transcription and protein
synthesis (e.g. actinomycin D and cycloheximide)
are ineffective to block those rapid effects (6, 7).
Rapid in vitro effects of aldosterone on electrolyte
and volume regulation, on the activity of the
Na+/H+-exchanger and intracellular signaling in
lymphocytes and vascular smooth muscle cells have
been shown within minutes (8-10), which are proba-
bly transmitted by membrane receptors for steroids
(11, 12). In the meantime, rapid effects have been
reported for almost all steroids in various target tis-
sues and cell systems (6).
Rapid modulation of aldosterone plasma levels has
been recognized in studies investigating the effect
of orthostatic stress and physical exercise on circu-
lating hormones (13, 14). Such rapid changes in al-
dosterone plasma levels would be ineffective if ge-
nomic effects of steroids were their only mode of
Key-words: Aldosterone, skeletal muscle, MR-spectroscopy, oxidative
metabolism.
Correspondence: Prof. Martin Wehling, Institute of Clinical Pharmacology,
Faculty of Clinical Medicine, University of Heidelberg, Theodor Kutzer
Ufer 1-3, 68167 Mannheim, Germany.
E-mail: martin.wehling@kpha.ma.uni-heidelberg.de
Accepted January 26, 2001.
587

Aldosterone effects on skeletal muscle
action. Indeed, rapid in vivo actions of aldosterone
have been characterized in addition to genomic ef-
fects: aldosterone rapidly increases systemic vas-
cular resistance in man (15, 16), and suppresses
baroreceptor sensitivity in dog and man (17, 18).
Rapid modulation of skeletal oxidative metabolism
of human calf after isometric exercise by aldos-
terone (5) supports the assumption that aldosterone
directly modulates extrarenal targets via rapid non-
genomic mechanisms. Administration of aldos-
terone significantly modulates the level of muscular
phosphocreatine (PCr) after isometric muscle con-
traction within 8 min (5). Post-exercise steady state
levels of PCr are significantly increased above pre-
exercise levels. However, aldosterone does not sig-
nificantly affect PCr levels and intracellular pH dur-
ing contraction indicating that the anaerobic ener-
gy metabolism is not modified. Furthermore, the
time constant of PCr recovery after isometric con-
traction is not changed by aldosterone, which im-
plies that the rate of oxidative ATP formation in the
initial phase of recovery was not modulated.
The mechanisms of this rapid aldosterone-induced
effect on skeletal muscle metabolism are not com-
pletely understood. Thus, the aim of the present
study was to further characterize aldosterone-in-
duced effects on oxidative energy metabolism dur-
ing recovery after isometric contraction. This was
done by comparison of the metabolic responses of
skeletal muscle to normoxia and hypoxia. Hypoxia
was chosen to test the dependence of the effect
on oxygen supply and, thus, its relation to oxida-
tive vs non-oxidative metabolism. The study was
conducted to test if aldosterone interferes with mi-
tochondrial oxidative recovery or by affecting fiber
type recruitment of the skeletal muscle.
ticipants with metal implants or pacemakers [con-
traindications for magnetic resonance (MR) spec-
troscopy]. Physical examinations, 12-lead electro-
cardiograms and ergometric exercise stress testing
did not reveal any relevant physical abnormalities of
study participants. One male subject was excluded
from further analysis as he was not able to carry out
continuous isometric plantar flexion for 3 min, which
was monitored by a strain gauge force transducer.
Study medication
Aldosterone for iv administration in humans was a
gift from Parke-Davis (Lainate, Italy). Placebo (0.9%
NaCl; 1 ml) and verum (0.5 mg/ml aldosterone in
0.9% NaCl; 1 ml) were prepared under sterile con-
ditions and verified as pyrogen-free.
MR data acquisition and processing
MR spectra of the right calf were performed in a
Bruker Biospec (Bruker-Medizintechnik, Karlsruhe,
Germany), which provides a magnetic field strength
of 4.7 Tesla in a horizontal magnetic bore of 40-cm di-
ameter. The 1H/31P double tuned surface coil (5-cm
diameter) was placed under the right calf at its largest
diameter. Thirty-two scans were averaged within 10 s
for each spectrum. The resonance frequency of the
pulse was 81 MHz for 31P, and the flip angle was 60°
at the center of the coil. Vector size was 4 k and spec-
tral width 10,000 Hz. Spectra were corrected for par-
tial spin saturation and their relative proportions from
total phosphate ([PME]+[Pi]+[PCr]+[ATP]) were de-
termined. [PCr] in resting muscle was set to 100%.
Changes in the levels of phosphomonoesters [PME],
inorganic phosphate [Pi], and adenosine trisphos-
phate [ATP] were determined in relation to [PCr].
Absolute concentrations of ATP, PCr, and Pi were esti-
mated by multiplying the peak areas of their signals
with the factor obtained by setting the resting av-
erage peak area of (β-P)-ATP to an assumed abso-
lute ATP-concentration of 8.2 mmol/l in cell water
(5). Free concentration of ADP was calculated by
assuming an equilibrium constant of 1.66·109 (Keq)
for the creatine kinase reaction being in equilibrium
([ADP]=(0.74·[ATP]·([TCr]-[PCr]))/(Keq·10-pH·[PCr]); (19)).
Oxidative ATP production was characterized by the
time constant of [PCr] recovery after isometric exer-
cise, which was calculated using a monoexponential
regression model. The fitting function was deter-
mined for a time interval lasting from 10 s to 4 min
after isometric contraction. pH was determined from
the chemical shift of the Pi signal (δPi) relative to δPCr=0
ppm (pH=6.75+log (δPi-3.27)/(5.69-δPi) (20).
PATIENTS, MATERIALS AND METHODS
Ethical approval
The study protocol and the methods used were ap-
proved by the Ethics Committee of the Landesärz-
tekammer Nordrhein, Düsseldorf, Germany. The
study was performed in accordance with the Decla-
ration of Helsinki and the ICH-guidelines.
Study volunteers
Ten healthy volunteers (7 men and 3 women; mean
age 30.8 7.0 yr) were recruited from the German
Aerospace Research Establishment (DLR, Cologne,
Germany). Medical history was taken to exclude
chronic or acute diseases such as coronary artery
disease, hypertension, pulmonary diseases, malig-
nancies, infections, and endocrine or psychiatric dis-
orders. Special care was taken to exclude study par-
Isometric foot plantar flexion was standardized in a
specially designed pedal ergometer (5). The maxi-
mum value of 3 tests of 5-s exercise intermitted by
588

M. Christ, J. Zange, C.P. Janson, et al.
55-s recovery maximum voluntary isometric force
(MIF) of calf muscle was determined. The contrac-
tion force at the ball of the foot was measured con-
tinuously and displayed to the subjects.
uration was measured by pulse oximetry for 30 min
during the initial adaptation period at normoxic or
hypoxic conditions and for 2 min at the end of the
examination protocol after the MR-spectroscopic
measurements.
Study design
Laboratory methods
The study was designed as a prospective, double-
blind, placebo-controlled, randomized trial in cross-
over design. Study volunteers were randomly as-
signed to a sequence of four tests: two investigations
(aldosterone or placebo) were done under normox-
emic conditions [inspiratory oxygen fraction (FiO2)=
0.21], and two investigations under hypoxemic con-
ditions (FiO2=0.13; comparable to an environment at
about 3600 m above sea level). All experimental in-
vestigations were done under standardized condi-
tions at a controlled room temperature of 20 C. The
two tests each at normoxemia and hypoxemia were
done at least 3 days apart. A minimum interval of 5 h
was interponed between aldosterone and placebo
tests for washout of aldosterone, which has a plasma
half-life of less than 30 min after iv administration (5).
On each study day, two teflon catheters (18G veno-
flon; Braun, Melsungen, Germany) were placed in the
left and right antecubital vein, one for blood sam-
pling and the other for iv aldosterone/placebo ad-
ministration. Study participants were investigated in
supine body position with the upper body elevated
by 25°. The right foot was fixed on the support of a
calf muscle ergometer (5), which was adjusted at an
angle of 70°. Force measurement with a strain gauge
transducer was continuously displayed to the partic-
ipants, who were advised to adjust their efforts to
closely match the preset tension level.
The examination protocol started with a period of 30
min, during which the subjects adapted to the hori-
zontal position and to the inspired gas mixture. Blood
samples for laboratory tests and hormone profiles
were drawn and MR spectroscopy was started. After
obtaining baseline MR-spectra for 3 min, injection of
study medication followed. Three minutes after in-
jection, blood samples for measurement of plasma
aldosterone were obtained. Isometric exercise was
started one minute later. The participants performed
4 steps of isometric plantar flexion at 40% MIF for 3
min. The first 3 exercise steps were followed by 5 min
of recovery, the last recovery period was 10 min long.
Afterwards MR spectroscopy was stopped and blood
samples were drawn for determination of aldosterone
plasma concentrations at the end of the test.
Blood samples were drawn in sample tubes with or
without EDTA via an indwelling catheter. EDTA-
plasma samples for hormones were centrifuged at
3000 g for 20 min at 4 C (aldosterone) or room tem-
perature (renin), and immediately frozen until fur-
ther processing. Routine parameters of hematolo-
gy (EDTA-plasma) and biochemistry (serum) were
determined by standard tests in a BM/Hitachi 717
(distributor: Boehringer Mannheim, Germany).
Plasma renin and aldosterone levels were determi-
ned by commercially available radioimmunoassay
kits (Serono Diagnostika, Freiburg, Germany; ERIA
Diagnostics, Pasteur, France).
Statistics
Results are presented as means standard devia-
tion (SD). The primary interest for inference statistics
were the modulation of phosphocreatine concen-
trations and muscular pH by treatments (hypoxia vs
normoxia; aldosterone vs placebo). Sample size of
the trial was prospectively calculated only to com-
pare areas-under-the-concentration time curves
(AUC) of [PCr], [ADP], and [Pi] for the 4th contrac-
tion and recovery periods due to the longer recov-
ery periods of 10 min. Using the AUC-method of-
fers the possibility to compare sequential points of
a large data set with one statistical test. For sec-
ondary variables ([PME], [ATP]), individual mean val-
ues over given time intervals, such as the first 3 min,
and the last 6 min after the time indicated, were
calculated in a descriptive approach. The increasing
rate of PCr degradation during repeated isometric
contractions of plantar flexors (21) was calculated
by linear regression analysis of the PCr-levels at the
end of each contraction period, and the possible
modulation by aldosterone or hypoxia was tested
by the comparison of the regression coefficients.
Due to the small sample size the normal distribu-
tion of parameters could not be proven; therefore
an analysis of variance (ANOVA) with a factorial
model was not considered to be appropriate. Thus,
the null hypothesis of no aldosterone effect was
tested by the non-parametric signed-rank-test for
paired data using a commercially available software
package (SAS V. 6.12, SAS Institute, Cary, NC). P-
values <0.1 were regarded to be statistically no-
ticeable, p-values <0.05 were considered statisti-
cally significant.
A 2-lead electrocardiogram (Bruker Physiograd SM
785 NMR, Bruker Medizintechnik, Karlsruhe, Ger-
many) was continuously monitored throughout the
test. Breathing rates and blood pressure were de-
termined at 2-5-min intervals (Fig. 1). Oxygen sat-
589

Aldosterone effects on skeletal muscle
Table 1 - Clinical characteristics and laboratory parameters of
nificantly different from each other. Basal renin lev-
els were increased by ~15-30% during hypoxia
compared with values determined during normoxia
(p=0.01). Three and 33 min after intravenous ad-
ministration of aldosterone, plasma levels of al-
dosterone were significantly increased ~50-70-fold
(p=0.0001 vs baseline and placebo) and ~10-20-
fold (p=0.001 vs baseline and placebo), respec-
tively. Plasma aldosterone remained unchanged
after injection of placebo.
Although the plasma half-life of aldosterone is less
than 30 min (5), a possible carry-over effect of
treatment was evaluated: plasma aldosterone
concentrations at baseline were 186.0 36.7
(mean SD, hypoxia) and 166.2 55.4 (normoxia)
pg/ml in volunteers, who were given aldosterone
at period 1, while levels at baseline were
165.6 78.0 (hypoxia) and 185.0 59.3 pg/ml (nor-
moxia) at investigation 2 of the experimental day.
Volunteers on placebo first had aldosterone plas-
ma concentrations of 154.1 51.2 (hypoxia) and
121.0 19.8 pg/ml (normoxia) at period 1, and
150.6 48.2 (hypoxia) and 131.5 39.0 pg/ml (nor-
moxia) at period 2. The ratio of first/second ap-
plication was 1.32 (hypoxia) and 0.91 (normoxia)
for the sequence aldosterone-placebo, and 1.02
(hypoxia) and 1.08 (normoxia) for the sequence
placebo-aldosterone. A carry-over was therefore
not likely to occur.
the study volunteers.
Parameter
Mean SD
3/6
Female/Male
Age (yr)
30.8 7.0
Weight (kg)
75.3 13.2
73.1 11.9
128.0 17.7
78.1 9.3
Heart rate (beats/min)
Systolic blood pressure (mmHg)
Diastolic blood pressure (mmHg)
Na+ (mmol/l)
137.7 5.81
4.18 0.27
0.92 0.17
8.36 2.5
K+ (mmol/l)
Creatinine (mg/dl)
ASAT (U/l)
ALAT (U/l)
11.82 7.47
47.22 5.34
0.43 0.04
14.88 1.37
Albumine (g/l)
Hematocrit (l/l)
Hemoglobin (g/dl)
ALAT: alinine amino transferase, ASAT: aspartate amino transferase; K+:
serum potassium; Na+: serum sodium.
RESULTS
Clinical characteristics of study participants
Nine volunteers (3 women, 6 men; mean age
30.8 7.0 yr, range 18-45; mean body weight
75.3 13.2 kg) fulfilled the requirements for enroll-
ment in the study and were analyzed. Basic clini-
cal characteristics and laboratory parameters are
shown in Table 1. All values were within the nor-
mal range.
Hemodynamic parameters, safety parameters,
force-time integrals during isometric plantar
flexion
While systolic and diastolic blood pressure did not
change during the experimental protocol or be-
tween the different experimental groups (data not
shown), heart rate significantly increased during
hypoxia (73.4 13.8 vs 64.4 11.8 bpm; p<0.001
vs normoxia). Oxygen saturation was significantly
decreased during hypoxia (91.8 3.5, 82.7 3.6,
and 83.4 3.7% 1 min after breathing reduced oxy-
Plasma hormone levels during the experiments
Aldosterone and renin plasma levels at baseline as
well as aldosterone levels at three and 33 minutes
after injection of study medication are shown in
Table 2. Baseline plasma levels of aldosterone and
renin during normoxia and hypoxia were not sig-
Table 2 - Plasma concentrations of aldosterone and renin (mean SD).
Aldosterone
baseline (pg/ml)
Aldosterone 3 min
after injection (pg/ml)
Aldosterone 33 min
after injection (pg/ml)
Renin baseline
(μU/ml)
Normoxia
Placebo
162.7 56.8
147.4 48.5
174.3 81.3
145.2 52.3
16.8 6.6
25.5 9.1
Aldosterone
Hypoxia
10,157.2 3604.3
2733.4 1968.0
Placebo
156.8 59.5
167.4 46.7
161.9 75.9
132.9 48.7
22.2 7.3
Aldosterone
8073.0 2262.7
1768.1 662.7
29.0 13.5
590

M. Christ, J. Zange, C.P. Janson, et al.
-60 min
N·min; aldosterone: 1206 92 vs 1174 81 N·min for
Supine body position,
preparation of equipment,
adaptation
the 4th isometric contraction period).
Aldosterone effects on aerobic and anaerobic
metabolism of human calf muscle
Levels of [PCr] were significantly decreased dur-
ing exercise to ~75 5% at the end of the 1st con-
traction and to 50 15% at the end of the 4th con-
traction (Fig. 2A and B). Analysis of [PCr] decrease
according to sex demonstrated a decrease of [PCr]
to ~72 10% and 51 15% in men and to 66 5%
and 55 15% in women during the first and fourth
contraction, respectively. Injection of aldosterone
did not modulate the rate of PCr-degradation,
which gives an estimate of changed recruitment
of muscle fibers, during the four sequential iso-
metric contractions of the calf muscle vs placebo.
Muscular [PCr] breakdown during isometric con-
traction was significantly decreased during hy-
poxia (p=0.01) after administration of aldosterone,
while this difference was not significant in the
placebo group. Probably due to the small sample
size, no differences were found in the analysis of
interactions (aldosterone vs placebo during nor-
moxia vs hypoxia).
-30 min
-4 min
FiO2=0.21 or 0.13
Injection of study medication
0 min
Isometric contraction (1st)
Recovery
8 min
Isometric contraction (2nd
)
Recovery
16 min
24 min
Isometric contraction (3rd)
Recovery
Isometric contraction (4th)
Recovery
As the potential existence of rapid aldosterone ac-
tion on muscular metabolism should be shown in
both sexes, women were included in the study.
Recovery kinetics of [PCr] after isometric exercise
were not different between male and female sub-
jects (data not shown). However, injection of aldos-
terone induced an overshoot of [PCr] recovery af-
ter the fourth contraction period during normoxia
(Fig. 2A). Under normoxic conditions, AUC were
857.2 24.8 and 875.5 15.4%·min (p=0.019) in the
4th recovery period. Thus, aldosterone induced an
average increase of [PCr] by about 2.1% during the
4th recovery, respectively. In contrast, aldosterone
induced changes on [PCr] were not detectable dur-
ing hypoxemia (Fig. 2B).
Time constants of [PCr] recovery (Table 3) were
slightly increased by about 5.3 to 22.8% during hy-
poxia compared to normoxia; however, values dur-
ing hypoxia were only significantly different to nor-
moxic conditions after the 3rd contraction (p=0.05).
Recovery kinetics were not significantly modified
by the injection of aldosterone.
37 min
End of test
Fig. 1 - Experimental protocol of a study period. Study partici-
pants were investigated in supine body position with the upper
body elevated, the right foot was fixed on the support of a calf
muscle ergometer (5). A 2-lead electrocardiogram was continu-
ously monitored throughout the test, breathing rates and blood
pressure were determined at 2-5-min intervals. Oxygen saturation
(O2-saturation) was measured by pulse oximetry before (during
the 30-min adaptation period at normoxemic and hypoxemic con-
ditions) and after the magnetic resonance (MR) spectroscopic mea-
surements for safety reasons. 31P-MR-spectra were continuously
recorded during four repetitive isometric contractions. Placebo or
aldosterone were given during hypoxia or normoxia as indicated.
gen, before and 45 min after injection of study
medication; p<0.001), while during normoxia oxy-
gen saturation was not changed during the whole
experimental protocol (97.1 1.2, 97.1 1.4, and
97.5 1.1%).
Isometric exercise was performed by all nine subjects
as scheduled. Force-time integrals of each contrac-
tion during hypoxia and normoxia were not signifi-
cantly different (placebo: 1214 90 vs 1202 79
Pi-levels were significantly increased from 11.3 0.7
to 32.9 2.6 and 51.6 4.6 mmol/l during the 1st and
4th contraction at normoxemic conditions. Con-
traction-induced increases of [Pi] were not modu-
lated by aldosterone or hypoxia. Hypoxia induced
a significant undershoot of Pi during recovery peri-
ods after all isometric contractions (AUC of the 4th
Pi recovery 88.9 5.1 for normoxia vs 72.6 5.7
591

Aldosterone effects on skeletal muscle
Injection of study
medication
A
110
100
90
ꢀ=PCr
80
70
60
p=0.055
p=0.04
p=0.019
50
ꢁ Placebo ꢂ Aldosterone
Injection of study
medication
110
100
90
B
Fig. 2- Mean phosphocreatine (PCr)
levels (percentage of initial values av-
eraged for 3 min before injection) in
calf muscles of healthy study volun-
teers determined by 31P-magnetic res-
onance (MR)-spectroscopy. At the time
indicated, aldosterone (ꢂ; 0.5 mg al-
dosterone in 0.9% NaCl) or placebo
(ꢁ; 0.9% NaCl) was given iv. Spectra
were recorded during 4 repetitive iso-
metric contractions followed by rest
during normoxia (FiO2=0.21; A) and
hypoxia (FiO2=0.13; B). Area-under-
curves (AUC) of phosphocreatine lev-
els after the first, third and fourth iso-
metric contraction were different be-
tween aldosterone and placebo tests
during normoxia (no.=9), but not dur-
ing hypoxia (no.=9). Representative er-
ror bars are given at the end of base-
line, and before and at the end of each
contraction interval.
80
70
60
50
0
5
10
15
20
25
30
35
40
Time (min)
covery period after the 4th contraction by aldos-
terone vs placebo during normoxia (AUC: 92.2 9.9
vs 100.6 10.6 μmol/l·min) and during hypoxia
(AUC: 112.2 19.6 vs 105.3 11.8 μmol/l·min) (Fig.
3). Concentrations of ATP and phosphomonoesters
(PME) were not significantly different during the ex-
perimental protocol as displayed in Table 4.
Injection of aldosterone did not change the time
course of changes in intracellular pH vs placebo
(Fig. 4) during normoxic or hypoxic conditions (da-
ta not shown).
mmol/l·min; p=0.02]. AUC of Pi-recoveries after
contractions displayed a significant undershoot in
the aldosterone vs placebo group during normox-
ia (p=0.05 for the 4th contraction), while no differ-
ences were evident during hypoxia. Muscular [Pi]
during hypoxia was significantly increased in the al-
dosterone group up to 116.6 8.2% (relative to val-
ues during normoxia; p=0.04) during the 4th con-
traction, while these differences were not evident
in the placebo group.
Muscular ADP-levels were not changed in the re-
592

M. Christ, J. Zange, C.P. Janson, et al.
Table 3 - Time constants of phosphocreatine (PCr) recovery (τ) of
study subjects after injection of placebo or aldosterone during
each interval of all experimental periods (normoxia vs hypoxia).
significant differences in contraction-induced de-
creases of PCr during normoxia and hypoxia are
observed after administration of placebo;
4) these effects occur at nanomolar blood concen-
trations of aldosterone.
Normoxia
Hypoxia
Placebo Aldosterone Placebo Aldosterone
The aldosterone-induced increases of PCr in the re-
covery period after isometric exercise and inverse
changes of Pi seen here confirm main results ob-
tained in the subgroup analysis of the previously
published study (5) supporting the hypothesis that
aldosterone modulates oxidative metabolism of hu-
man calf muscle. While muscular ATP content is
maintained during and after isometric contraction,
aldosterone enhances the increase of PCr in the re-
covery period after isometric exercise. In addition,
an aldosterone-induced undershoot of Pi has been
detected.
Two hypotheses might be proposed: aldosterone
modulates respiratory control at the cellular level
or it affects the type of muscle fiber recruitment.
Kushmerick et al. reported an overshoot of PCr in
cat soleus muscle compared to the lack of PCr
overshoot in the cat biceps muscle (19). As the cat
soleus muscle is primarily composed of slow-
twitch oxidative cells (22), aldosterone may switch
the type of muscle fiber recruitment in human calf
muscle from fast-twitch oxidative-glycolytic cells
to slow-twitch oxidative cells. Since distinct mus-
cle cell types differ in their mechanical and
metabolic characteristics reflecting different mi-
tochondrial contents and protein patterns (23), a
1st period
2nd period
3rd period
4th period
28.8 1.1 27.4 1.6
25.3 1.3 23.9 1.5
24.5 1.2 25.4 1.1
25.6 1.2 27.9 1.1
29.2 1.5 28.1 2.1
25.4 1.6 28.1 1.6
27.8 1.4* 28.1 1.4
27.1 1.6 29.0 1.9
Means SD; *p=0.05 vs respective level during normoxia.
DISCUSSION
The main results of the study are the following:
1) levels of PCr and Pi during recovery after iso-
metric exercise are significantly affected by al-
dosterone within minutes. Hypoxia (FiO2=0.13)
completely blocks aldosterone-induced effects
on oxidative metabolism of human calf muscle.
An aldosterone-induced modulation of the time
constant of the increase of PCr in the recovery
period after isometric exercise, which is a mark-
er of the aerobic ATP synthesis rate, cannot be
detected;
2) skeletal muscle pH is not affected by aldos-
terone;
3) exercise-induced degradation of muscular PCr is
significantly lower during hypoxia vs normoxia
after administration of aldosterone, while no
ꢁ Placebo ꢂ Aldosterone
45
40
35
30
25
20
15
10
5
Injection of study
medication
Fig. 3 - Muscular ADP in calf muscles of
healthy study volunteers determined by
31P- magnetic resonance (MR)-spec-
troscopy. At the time indicated, aldos-
terone (ꢂ; 0.5 mg in 0.9% NaCl) or place-
bo (ꢁ; 0.9% NaCl) were given iv. Spectra
were recorded during four repetitive iso-
metric contractions followed by rest dur-
ing normoxia (FiO2=0.21) and muscular
ADP levels calculated (see Methods sec-
tion). Muscular ADP was not significantly
changed in the aldosterone group vs
placebo during normoxemia and hypox-
emia (data not shown). Representative er-
ror bars are given at the end of baseline,
0
5
10
15
20
25
30
35
40
and before and at the end of each con-
traction interval. PCr: phosphocreatine.
Time (min)
593

Aldosterone effects on skeletal muscle
Table 4 - Muscular ATP and phosphomonoester (PME) at baseline, during and after the 4th contraction. Individual mean values over
given time intervals (means SD).
Baseline (first 3 min)
(% of initial PCr)
During 4th contraction
(3 min) (% of initial PCr)
After 4th contraction
(last 6 min) (% of initial PCr)
ATP
Normoxia
Placebo
26.3 3.3
25.9 2.8
25.4 3.0
25.3 3.6
26.3 3.3
25.9 3.0
Aldosterone
Hypoxia
Placebo
26.4 2.4
26.5 3.1
25.7 2.2
25.5 2.8
26.4 2.2
26.3 3.2
Aldosterone
PME
Normoxia
Placebo
3.9 1.4
3.6 1.9
7.6 2.1
6.7 2.5
6.2 2.8
5.2 2.6
Aldosterone
Hypoxia
Placebo
3.1 0.6
2.8 1.0
8.7 2.3
7.7 3.0
5.5 2.0
5.2 1.8
Aldosterone
PCr: phosphocreatine.
of H+ due to differences in metabolic efficiencies
and aerobic or anaerobic capacities. Such changes
were not observed. Furthermore, the increasing
rate of PCr degradation after repetitive isometric
contractions, which gives an estimate for the
change of the nervous recruitment pattern of plan-
change of the recruitment of muscle fiber types
would explain the aldosterone-induced PCr over-
shoot in the recovery period after isometric exer-
cise. However, a change of fiber type recruitment
during contraction by aldosterone would induce
alterations in PCr consumption and accumulation
Injection of study
ꢁ Placebo ꢂ Aldosterone
medication
7.15
7.10
7.05
7.00
6.95
6.90
Fig. 4 - Muscular pH in calf mus-
cles of healthy study volunteers
determined by 31P- magnetic
resonance (MR)-spectroscopy. At
the time indicated, aldosterone
(ꢂ; 0.5 mg in 0.9% NaCl) or
placebo (ꢁ; 0.9% NaCl) were
given iv. Spectra were recorded
during 4 repetitive isometric con-
tractions followed by rest during
normoxia (FiO2=0.21). Muscular
pH was not significantly changed
in the aldosterone group vs
placebo during normoxemia and
hypoxemia (data not shown).
Representative error bars are
given at the end of baseline, and
0
5
10
15
20
25
30
35
40
before and at the end of each
contraction interval.
Time (min)
594

M. Christ, J. Zange, C.P. Janson, et al.
tar flexors (23), appears to be unchanged by al-
dosterone treatment. Thus, this hypothesis seems
unlikely to be relevant.
undershoot of Pi was completely blocked by
acute hypoxemic conditions. Hypoxemia does not
directly affect metabolic patterns of skeletal mus-
cle upon isometric exercise at 40% maximum vol-
untary force vs normoxia, as reported previously
(25). There was a significantly higher amount of
PCr degradation by isometric contraction during
hypoxia vs normoxia in the aldosterone group,
while PCr degradation was not changed in the
placebo group. This means that higher amounts
of energy are needed in the aldosterone group
during hypoxia to exert the same level of force
pointing to an aldosterone-induced imbalance of
ATP utilization. Thus, we speculate that hypoxia
masks the transient imbalance of the ATP syn-
thesis rate and the rate of ATP utilization induced
by aldosterone. This increased incorporation of
Pi in PCr would also give an explanation for the
undershoot of Pi, because the major alternative -
Pi trapping in the glycogenolytic pathway (20, 26)
- was not altered by aldosterone. The mecha-
nisms linking oxygen-supply to aldosterone-in-
duced imbalance of ATP synthesis and utilization
rate are yet unknown. This imbalance may be ex-
plained by an altered sensitivity of the mitochon-
dria to control by ADP concentrations. Increases
of intracellular Ca2+, which has been demonstrat-
ed to be induced by aldosterone in vascular
smooth muscle cells within minutes (27) may al-
ter sensitivity of the mitochondria to pyridine nu-
cleotide (including ADP) control as demonstrat-
ed recently (28). Thus, a possible hypothesis
might be that the setpoint of respiratory sensitiv-
ity to the ADP concentration (28), which is
changed through rapid aldosterone effects dur-
ing normoxia, is altered by hypoxia.
The time elapsing until aldosterone-induced effects
on PCr and Pi after isometric contraction of plantar
flexors become visible is consistent with the time
frame of non-genomic steroid action, which is main-
ly characterized by a short delay (<10 min) (28).
Thus, the findings in the present study strongly sup-
port the existence of non-genomic aldosterone ef-
fects in vivo. Rapid aldosterone effects are ob-
served in the presence of endogenous hormone
and indicate that the rapid effector is sensitive to
exogenous doses of aldosterone. High doses of al-
dosterone applied in the present study were used
to obtain maximal effects, as small effects were ex-
pected as seen in in vitro observations. Still, the
concentrations of aldosterone applied here are in
the nanomolar range. These comparably high con-
centrations are yet far from concentrations at which
unspecific action would be suspected to occur (>10
μmol/l) (6).
Aldosterone may directly influence respiratory
control to induce PCr overshoot, which must be
discussed considering the combined (Lohmann)
creatine kinase (CK) reaction (H++PCr–+Mg-
ADP–↔ Mg–ATP–+Cr). Following parameters may
contribute to increase [PCr] over the initial levels at
rest: 1) muscular Mg2+ concentration; 2) the cellu-
lar pH; and 3) the mitochondrial phosphorylation
potential reflected by the ATP/ADP ratio or the
ADP content at constant ATP levels. An aldos-
terone-induced decrease in 1) the Mg2+ concen-
tration would have shifted the position of the equi-
librium of the CK reaction to the left, raising [PCr]
and [ADP] and possibly inducing a transient in-
crease in respiration that would further decrease
[Pi]. However, such changes were not observed in
the chemical shifts of the ATP signals (data not
shown).
Aldosterone may influence creatine kinase reaction
via 2) an impact on cytosolic pH. Aldosterone rapid-
ly stimulates the Na+/H+ antiporter activity in vas-
cular smooth muscle cells (9) resulting in cellular al-
kalinization. However, aldosterone-induced alter-
ations of muscular pH were not detected in the re-
covery period after isometric exercise between the
experimental groups (Fig. 4 and 5). In addition, ex-
cercise-induced increase of muscular pH would
drive the CK-reaction to the right suggesting a de-
crease of muscular PCr. Since the change in pH was
greater in the cat biceps (where no PCr overshoot
was detectable) than in the soleus muscle of the cat
(19), a pH and Na+/H+ antiporter-dependent mech-
anism of aldosterone-induced overshoot of PCr is
unlikely.
The 3) overshoot of PCr reflects an increase of the
phosphorylation potential. Assuming that oxygen
supply is not limiting, the [ATP]/[ADP] ratio is be-
lieved to be the predominant factor in the regula-
tion of oxidative phosphorylation during recovery
in skeletal muscle (24). Although a trend to an un-
dershoot of ADP-recovery was seen, the rate of PCr
recovery, which was measured by the time constant
of PCr recovery after isometric contraction (τ), was
not changed by aldosterone and/or hypoxia sug-
gesting that oxygen supply was not limiting in the
recovery period after isometric exercise in any
phase of the study. Thus, we hypothesize that ei-
ther the ATP turnover was reduced via an imbal-
ance of the ATP-synthesis or utilization rate, or the
regulation of ATP synthesis was altered by aldos-
terone.
The aldosterone-induced overshoot of PCr and
595

Aldosterone effects on skeletal muscle
9. Christ M., Meyer C., Sippel K., Wehling M.
Rapid aldosterone signaling in vascular smooth mus-
cle cells: involvement of phospholipase C, diacyl-
glycerol and protein kinase C alpha.
In summary, aldosterone rapidly modulates mus-
cular PCr and Pi changes in the recovery periods
after isometric contraction. These effects are
blocked by hypoxia. The presented data are fully
compatible with earlier in vitro and in vivo obser-
vations on non-genomic aldosterone action in
man, and suggest that aldosterone probably re-
duces ATP turnover by causing an oxygen-de-
pendent imbalance of ATP utilization and ATP syn-
thesis rate.
Biochem. Biophys. Res. Commun. 1995, 213: 123-129.
10. Christ M., Günther A., Heck M., Schmidt B.M.,
Falkenstein E., Wehling M.
Aldosterone, not estradiol, is the physiological ag-
onist for rapid increases in cAMP in vascular smooth
muscle cells.
Circulation 1999, 99: 1485-1491.
11. Meyer C., Christ M., Wehling M.
Characterization and solubilization of novel al-
dosterone binding proteins in porcine liver mi-
crosomes.
ACKNOWLEDGMENTS
We kindly appreciate expert technical assistance by Mrs.
Elke Kirsch. The study was supported by a grant of the
Bundesministerium für Bildung, Forschung und Wissenschaft
(01EC9407/8).
Eur. J. Biochem. 1995, 229: 736-740.
12. Wehling M., Eisen C., Christ M.
Aldosterone-specific membrane receptors and rapid
non-genomic actions of mineralocorticoids.
Mol. Cell. Endocrinol. 1992, 90: C5-C9.
13. Hinghofer-Szalkay H.G., Vigas M., Sauseng-Fellegger
G., Konig E.M., Lichardus B., Jezova D.
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