J Appl Physiol
89: 1033–1038, 2000.
Endurance training does not alter the level of
neuronal nitric oxide synthase in human skeletal muscle
U. FRANDSEN, L. HÖFFNER, A. BETAK, B. SALTIN, J. BANGSBO, AND Y. HELLSTEN
Department of Human Physiology, Copenhagen Muscle Research Centre,
University of Copenhagen, DK-2100 Copenhagen Ø, Denmark
Received 3 February 2000; accepted in final form 26 May 2000
(NOS), an enzyme that synthesizes nitric oxide (NO) from L-arginine, exists in several isoforms. In human skeletal muscle, endothelial
NOS (eNOS) is located in the vascular endothelium
and neuronal NOS (nNOS) is located in the sarcolemma and cytosol of the muscle fibers, in apparent
association with mitochondria (6). NOS has been
shown to be of importance in the regulation of several
events in skeletal muscle tissue. There is evidence
supporting a role for NO in the regulation of muscle
blood flow, at least at rest and in recovery from exercise
(4, 15). An interaction between NO and superoxide
radicals in the regulation of muscle contractile force
has been demonstrated (12), and a role for NO has also
been implicated in the regulation of mitochondrial respiration (17) as well as in insulin-mediated glucose
uptake (14, 21). The importance of NOS for skeletal
muscle function is clear, and enhanced contractile activity could be expected to result in an elevated level of
the enzyme. In congruence with this hypothesis, Reiser
et al. (13) showed that chronic in vivo stimulation of
rabbit muscle for 3 wk induced a marked increase in
nNOS expression and activity in parallel with a fastto-slow fiber-type transformation. Similarly, NOS expression was found to increase in rat skeletal muscle,
after 8 wk of treadmill running (1) and in muscle cell
cultures after 2 days of contractile activity induced by
electrical stimulation (20). It is not known, however,
whether endurance training in humans may alter the
level of nNOS in skeletal muscle or whether such an
alteration is restricted to oxidative fibers.
In models of isolated skeletal muscle, it has been
shown that NO is formed at a higher rate during
contraction compared with at rest (1, 20). The amount
of NO formed is frequently assessed by measurements
of the hydration products of NO in aqueous solutions,
nitrite and nitrate. In previous studies on NO formation in skeletal muscle, nitrite and nitrate have been
measured in medium surrounding the isolated muscle
preparations (2, 20); however, nitrite and nitrate levels
have not previously been measured directly in the
muscle tissue. Such measurements performed on muscle biopsies before and after an exercise bout, for a
trained and an untrained muscle group, could provide
an indication of the in vivo formation of NO in humans
and whether training affects NO production.
The primary hypothesis of the present study was
that endurance training of sedentary human subjects
would result in a higher level of skeletal muscle nNOS
protein and activity and that this increase mainly
would occur in the cytosol of type I fibers. Muscle
biopsies obtained before and after training were analyzed for nNOS protein, NOS activity, distribution of
nNOS and eNOS, and nitrite and nitrate levels before
Address for reprint requests and other correspondence: Y. Hellsten, Dept. of Human Physiology, Copenhagen Muscle Research
Centre, Univ. of Copenhagen, Universitetsparken 13, DK-2100
Copenhagen Ø, Denmark (E-mail: [email protected]).
The costs of publication of this article were defrayed in part by the
payment of page charges. The article must therefore be hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
nitric oxide synthase activity; immunohistochemistry; nitrite; nitrate; exercise; endothelial nitric oxide synthase
NITRIC OXIDE SYNTHASE
http://www.jap.org
8750-7587/00 $5.00 Copyright © 2000 the American Physiological Society
1033
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Frandsen, U., L. Höffner, A. Betak, B. Saltin, J.
Bangsbo, and Y. Hellsten. Endurance training does not
alter the level of neuronal nitric oxide synthase in human
skeletal muscle. J Appl Physiol 89: 1033–1038, 2000.—The
effect of endurance training on neuronal nitric oxide synthase (nNOS) content and distribution in muscle was investigated. Seven male subjects performed 6 wk of one-legged
knee-extensor endurance training (protocol A). Muscle biopsies, obtained from vastus lateralis muscle in the untrained
and the trained leg, were analyzed for nNOS protein and
activity as well as immunohistochemical distribution of
nNOS and endothelial nitric oxide synthase (eNOS). Muscle
biopsies were also obtained from another seven male subjects
before and after 6 wk of training by endurance running
(protocol B) and analyzed for nNOS protein. No difference
was found in the amount of nNOS protein in the untrained
and the trained muscle either with protocol A or protocol B
(P ⬎ 0.05). In protocol A, the activity of nNOS was 4.76 ⫾
0.56 pmol 䡠 mg protein⫺1 䡠 min⫺1 in the control leg, and the
level was not different in the trained leg (P ⬎ 0.05). nNOS
was present in the sarcolemma and cytosol of type I and type
II muscle fibers, and the qualitative distribution was similar
in untrained and trained muscle. The number of eNOS immunoreactive structures and the number of capillaries per
muscle fiber were higher (P ⬍ 0.05) after training than
before. The present findings demonstrate that, in contrast to
findings on animals, nNOS levels remain unaltered with
endurance training in humans. Evidence is also provided
that endurance training may increase the amount of eNOS,
in parallel with an increase in capillaries in human muscle.
1034
ENDURANCE TRAINING AND HUMAN MUSCLE nNOS
and after exercise. The training protocol, utilizing onelegged knee-extensor exercise, was chosen to allow for
training with a well-defined muscle group and to have
a matching untrained muscle for control tissue. In
addition, for comparative purposes, the effect of training with a larger muscle mass, and thus a greater
hormonal response, on muscle nNOS levels was also
examined in another group of subjects performing running training for a similar period.
MATERIALS AND METHODS
Downloaded from http://jap.physiology.org/ by 10.220.33.6 on April 6, 2017
Protocol A. Seven healthy male subjects with a mean age of
24 ⫾ (SE) 1 yr (range 20–32 yr), mean body weight of 76.1 ⫾
2.7 kg (62.0–85.0 kg) and mean maximum oxygen uptake
(V̇O2 max) of 45.4 ⫾ 1.9 ml 䡠 kg⫺1 䡠 min⫺1 (39.2–54.8
ml 䡠 kg⫺1 䡠 min⫺1) participated in the study. The subjects were
informed of any risks and discomfort associated with the
experimental procedures before giving their informed consent to participate. The study was approved by the Local
Ethical Committee of Copenhagen and Fredriksberg. The
training was performed three times per week for 6 wk and
consisted of 1-min bouts of one-legged knee-extensor training, separated by 30 s of rest, performed repeatedly for 1 h.
The workload had been set in previous tests to result in
exhaustion after ⬃1 h. The mean work rate for the subjects
was increased successively during the training period so that
the work rates at the beginning and at the end of the training
period were 51.7 ⫾ 0.7 and 64.6 ⫾ 2.8 W, respectively.
Muscle biopsy samples were obtained with the needlebiopsy technique under local anesthetic. Samples were obtained from the trained leg before the training and from the
trained and the control leg 24 h after the last training
session. The biopsies were divided into two portions, one of
which was frozen directly in liquid nitrogen and the other
mounted and frozen in isopentane cooled in liquid nitrogen
for immunohistochemistry.
A performance test was conducted several days before the
first training session and 2 days after the last training
session. Subjects performed graded one-legged knee-extensor
exercise to exhaustion in which the work rate initially was
set at 10 W and was then increased by 10 W every 4 min until
40 W, and thereafter by 6 W every 4 min until the subjects
could no longer maintain the kicking frequency.
The subjects also performed two 20-min exercise bouts at
30 W after the training period, one with the control leg and
one with the trained leg in randomized order. The bouts were
separated by 30 min. Muscle biopsies were obtained from the
vastus lateralis muscle before and immediately after the
exercise. The biopsies were immediately frozen in liquid
nitrogen and stored at ⫺80°C until analysis for nitrite and
nitrate. Before analysis, the muscle biopsies were freezedried and dissected free from visible connective tissue, fat,
and blood.
Protocol B. Eight healthy male subjects with a mean
age of 23 ⫾ 1 yr (20–28 yr), mean body weight of 80.0 ⫾ 3.2
kg (66.0 – 93.5 kg), and mean V̇ O 2 max of 49.4 ⫾ 1.6
ml 䡠 kg⫺1 䡠 min⫺1 (43.3–55.1 ml 䡠 kg⫺1 䡠 min⫺1) participated in a
6-wk training program. The first 3 wk of training were
performed three times per week, and, during the next 3 wk,
training was performed four times per week. The training
sessions consisted of endurance running at either ⬃65% of
V̇O2 max for 30–45 min or three 8- to 10-min intervals, interspersed by 3 min of rest, at the highest possible intensity that
could be maintained through all three bouts.
Muscle biopsy samples were obtained before the training
and 24 h after the 6 wk of training. Several days before and
2 days after the final training session, the subjects performed
an intermittent shuttle run test to assess endurance performance. Performance was determined as time to exhaustion.
Western blotting. Western blot analysis was performed on
freeze-dried, dissected muscle biopsy samples. The proteins
were separated according to their molecular mass by SDSPAGE. Each sample (15 ␮g) was run in duplicate on two
identical gels. Immunoblotting was conducted by using polyvinylidene difluoride membranes (Millipore, Bedford, MA),
and the transfer was carried out for 1 h at 225 mA/membrane
and 100 V.
The membranes were blocked with 5% nonfat dried milk in
20 mM Tris, 500 mM NaCl, and 1% Tween-20 (pH 7.5) for 1 h,
and the membranes were subsequently incubated overnight
at 4°C with the primary nNOS antibody (N31030, Transduction Laboratories, Lexington, KY). After washing, antibody
binding was visualized by incubation with a secondary antibody coupled to alkaline phosphatase, followed by detection
with enhanced chemiluminescence (ECL, Amersham, Buckinghamshire, UK). The blots were quantified with densitometric scanning by using a densitometer (Molecular Dynamics Storm 840) with digital analysis software (ImageQuaNT).
The densities of the protein bands were quantitated as local
average over background, standardized to the local average
observed for a rat brain standard so that the different gels
could be compared.
NOS activity. NOS activity was determined in freezedried, dissected biopsies from protocol A with slight modification of the citrulline assay (3). Briefly, the reaction was
started by incubation of 10 ␮l samples (⬇200 ␮g) in a buffer
consisting of 32.25 mM Tris 䡠 HCl (pH 7.4). 3.75 ␮M BH4, 0.63
␮M flavin adenine mononucleotide, 1.15 mM NADPH, 3.5
␮M L-[14C]arginine, 0.7 mM CaCl, 0.1 ␮M calmodulin, and
distilled water to a final volume of 130 ␮l. The reaction was
carried out at 37°C for 20 min, and the reaction was terminated by the addition of 400 ␮l of an ice-cold stop buffer (50
mM HEPES and 5 mM EDTA, pH 5.5). Then, 200 ␮l of resin
buffer consisting of 1 g AG 50w-8x (Na⫹ form) resin in 1 ml
of 50 mM HEPES buffer (pH 5.5) were added to the solution,
and the resin-containing incubates were transferred to centrifuge tube filters (SPIN-X, Costar) and centrifuged for 1
min at 15,000 g to eliminate excess L-[14C]arginine. Scintillation liquid was added to the eluates, and the eluates were
counted in a Packard 2300TR liquid scintillation counter.
The enzyme activity is expressed as picomoles of L-citrulline
produced per milligram of protein per minute.
Vmax and Km were determined according to the MichaelisMenten equation from measurements of the NOS activity at
concentrations of L-arginine ranging from 0.014 to 7.51 ␮M.
Immunohistochemistry. The immunohistochemical analysis was performed on cross sections (8 ␮m) cut from frozen
muscle samples obtained from the experimental leg before
and after training. For the staining of nNOS, the sections
were air dried for 5 min, blocked in 1% BSA, and incubated
with the primary antibody (B220–1, Eurodiagnostica,
Malmø, Sweden) at room temperature for 90 min. Binding
was visualized by incubation with a secondary antibody coupled to biotin, followed by an streptavidin-FITC complex.
For the staining of eNOS, the sections were fixed with 4%
formaldehyde for 5 min. Sections were blocked in 1% BSA
and incubated with primary antibody (N30020, Transduction
Laboratories) for 90 min at room temperature. For the staining of capillaries, the sections were air dried and stained for
ENDURANCE TRAINING AND HUMAN MUSCLE nNOS
1035
Fig. 1. Relative neuronal nitric oxide synthase (NOS) protein content determined by Western blotting (A) and NOS activity determined by the citrulline assay (B) in human muscle biopsy samples
obtained from vastus lateralis muscle in the experimental leg before
(Pre tr) and in the experimental (Post tr) and the control leg (Post
utr) after 6 wk of 1-legged knee-extensor endurance training. Values
are means ⫾ SE. Neuronal NOS protein content values are expressed relative to a rat brain standard.
45 min with primary antibody CD31 (M0823, DAKO A7S,
Glostrup, Denmark). Binding was visualized as for nNOS.
Type I fibers were identified with a monoclonal antibody to
myosin heavy chain I (M-8421, Sigma Chemical, St. Louis,
MO).
Muscle nitrite and nitrate. Freeze-dried, dissected muscle
biopsy samples were homogenized in doubly distilled water
at a concentration of 1 mg/300 ␮l and centrifuged at 10,000 g
for 20 min. The supernatant was saved and centrifuged at
100,000 g for 15 min. The supernatant was then filtered
Fig. 2. Distribution of neuronal NOS in cross sections of untrained
(a) and endurance-trained (b) human skeletal muscle. Note the
stronger staining of cytosol and sarcolemma in type I muscle fibers
(indicated by *). Original magnification ⫻400.
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through filters with a molecular mass cutoff of 30 kDa (Millipore, Bedford, MA). Nitrite and nitrate concentrations were
measured with chemiluminescence reaction with ozone. Injection volumes of 25 ␮l for nitrate and 10 ␮l for nitrite plus
nitrate were used. (NOA 280, Sievers, Boulder, CO; Ref. 7).
Citrate synthase activity. The activity of citrate synthase
was measured in freeze-dried, dissected muscle biopsy samples obtained from the control and the experimental legs
after 6 wk of training. The assay was conducted at 25°C and
was performed according to the method described by Lowry
and Passonneau (11).
Protein determination. Protein was determined in triplicate by using a colorimetric assay with bichinchoninic acid
(18). Albumin was used as standard.
Statistics. Data are presented as means ⫾ SE. In comparing group means for the experimental leg before and after
training, a paired Student’s t-test was used, whereas group
means for the untrained and trained legs were compared
with an unpaired Student’s t-test. Group means for nitrite
and nitrate in the untrained and the trained leg before and
after exercise were compared by using a two-way analysis of
variance. Differences were considered statistically significant
when P ⬍ 0.05.
1036
ENDURANCE TRAINING AND HUMAN MUSCLE nNOS
Table 1. Distribution of nNOS and eNOS in
untrained and endurance-trained
human skeletal muscle
nNOS
Untrained
Trained
eNOS
Untrained
Trained
Type I
Sarcolemma
Type I
Cytosol
Type II
Sarcolemma
Type II
Cytosol
⫹⫹⫹
⫹⫹⫹
⫹⫹
⫹⫹
⫹⫹
⫹⫹
⫹
⫹
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
Microvessels
⫹⫹
⫹⫹
nNOS, neuronal nitric oxide synthase; eNOS, endothelial nitric
oxide synthase. ⫺, Absence of staining; ⫹, faint staining; ⫹⫹, intermediate staining; ⫹⫹⫹, distinct staining.
RESULTS
Fig. 3. Nitrite (open bars) and nitrate (solid bars) concentrations in
untrained (Utr) and trained (Tr) muscle at rest and after exercise
(ex). Values are means ⫾ SE. dw, Dry weight.
DISCUSSION
The present study examined the effect of endurance
training on the protein level, activity, and distribution
of nNOS in human skeletal muscle. The results demonstrate that, in contrast to findings on animals, endurance training of humans does not alter the muscle
Fig. 4. Neuronal NOS protein content in human muscle biopsy
samples obtained from vastus lateralis muscle before (Pre) and after
(Post) 6 wk of endurance running training. Values are means ⫾ SE
expressed relative to a rat brain standard.
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Protocol A. Performance, as assessed by the final
work rate that the subjects exercised at in a graded
one-legged knee-extensor test to exhaustion, was improved from 54.9 ⫾ 4.2 to 66.3 ⫾ 2.7 W (P ⬍ 0.05).
After the 6 wk of training, the nNOS protein level in
the experimental leg was not different from pretraining values or from values of the control leg (P ⬎ 0.05;
Fig. 1A).
The calculated Vmax for NOS in skeletal muscle homogenates was 102 pmol 䡠 mg protein⫺1 䡠 min⫺1, and the
Km for L-arginine was 1.4 ␮M. The NOS activity level
in the experimental leg before training (4.76 ⫾ 0.56
pmol 䡠 mg protein⫺1 䡠 min⫺1) was similar to that of the
experimental leg and that of the control leg after training (P ⬎ 0.05; Fig. 1B).
Immunohistochemical analysis of muscle samples
obtained from the untrained muscle revealed positive
nNOS staining in the sarcolemma and in the cytosol of
the muscle fibers (Fig. 2). The staining of both the
sarcolemmal region and of the cytosol was stronger in
the type I fibers compared with type II fibers (Table 1).
There was no qualitative difference in the distribution
of nNOS staining in muscle samples obtained from the
experimental leg before and after training.
Positive eNOS staining was observed in the endothelium of microvessels. There was a higher number of
eNOS-immunoreactive structures in the experimental
leg with training (an increase from 2.03 ⫾ 0.15 to
2.28 ⫾ 0.4 structures/muscle fiber; P ⬍ 0.05). There
was also an increase in the number of CD31-immunoreactive microvessels in the experimental leg with
training, from 2.67 ⫾ 0.5 to 2.97 ⫾ 0.64 vessels/muscle
fiber (P ⬍ 0.05).
The concentrations of nitrite and nitrate were similar in the control and the trained leg at rest (P ⬎ 0.05;
Fig. 3). After 20 min of submaximal, one-legged kneeextensor exercise, the levels of nitrite and nitrate were
unchanged (P ⬎ 0.05) in both the control and the
trained legs.
The activity of citrate synthase in muscle was significantly higher (P ⬍ 0.05) in the trained leg (38.4 ⫾ 1.99
␮mol 䡠 min⫺1 䡠 g dry wt⫺1) than in the control leg (34.3 ⫾
1.6 ␮mol 䡠 min⫺1 䡠 g dry wt⫺1) after the 6-wk training
period.
Protocol B. Performance, as assessed by time to exhaustion in an intermittent shuttle-run test, was improved from 10.3 ⫾ 1.2 to 12.9 ⫾ 1.4 min (P ⬍ 0.01).
The relative amount of nNOS protein in the muscle
was similar before and after the 6 wk of training (P ⬎
0.05; Fig. 4).
ENDURANCE TRAINING AND HUMAN MUSCLE nNOS
do not support previous findings from animal studies in
vitro, which show an increase in nitrite and nitrate in
buffer surrounding contracting muscle (1, 20). The
reason for this latter apparent discrepancy is unclear
but could potentially be explained by the use of different experimental models.
In addition to the analysis of nNOS levels, an immunohistochemical analysis of eNOS was performed that
showed eNOS-positive staining in the endothelium of
microvessels. eNOS immunoreactivity was not observed in muscle fibers, a finding that contrasts with
the results from one of the first reports on the distribution of NOS in rodent muscle that show eNOS in the
cytosol in association with mitochondrial density (10).
A more recent study on rat diaphragm muscle has,
however, shown that the localization of eNOS is restricted to the vascular endothelial cells (5). The reason
for these dissimilarities remains unclear but could
potentially be related to the use of different antibodies
with differing antigen specificities.
In comparing untrained and trained muscle, the
number of eNOS-immunoreactive structures, tentatively identified as microvessels, per muscle fiber was
found to be significantly higher in the trained muscle,
as was the number of capillaries per muscle fiber. This
finding suggests that, when the number of capillaries
in the muscle is enhanced by endurance training, the
total amount of eNOS in the muscle increases.
In conclusion, the present study shows that endurance training in humans does not alter the protein
level, the activity, or the distribution of nNOS in skeletal muscle. Endurance training does, however, increase the number of eNOS-immunoreactive microvessels per muscle fiber in parallel with an increase in the
number of capillaries. Moreover, the levels of nitrite
and nitrate in untrained muscle are similar to those of
endurance-trained muscle and are not altered with
submaximal exercise.
We acknowledge the excellent technical assistance of Merete
Vannby, Inge-Lise Kring, and Karina Olsen. We also thank Jesper
Hauge and Peter Løwenstein for help with training the subjects.
The project was funded by Danish National Research Foundation
Grant 504-14, Team Denmark, and The Sports Research Council
(Idrættens Forskningsråd).
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