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1 Cardiovascular Division, Department of Internal Medicine, Institute of Clinical Medicine, 3 Department of Sports Medicine, Institute of Health and Sport Sciences, 4 Department of Environmental Medicine, Institute of Community Medicine, 2 Center for Tsukuba Advanced Research Alliance, University of Tsukuba, Tsukuba, Ibaraki 305-0006, Japan
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Cardiac myocytes
produce nitric oxide (NO). We studied the effects of intense exercise
on the expression of NO synthase (NOS) and the tissue level of nitrite
(NO2
)/nitrate (NO3) (i.e., NOx),
which are stable end products of NO in the heart. Rats ran on a
treadmill for 45 min. Immediately after this exercise, the heart was
quickly removed. Control rats remained at rest during the same 45-min
period. The mRNA level of endothelial NOS (eNOS) in the heart was
markedly lower in the exercised rats than in the control rats. Western
blot analysis confirmed downregulation of eNOS protein in the heart
after exercise. Tissue NOx level in the heart was significantly lower
in the exercised rats than in the control rats. The present study
revealed for the first time that production of NO in the heart is
decreased by intense exercise. Because NO attenuates positive inotropic
and chronotropic responses to 
1-adrenergic stimulation
in the heart, the decrease in cardiac production of NO by intense
exercise may contribute to the acceleration of increase in myocardial
contractility and heart rate during intense exercise.
nitric oxide; nitric oxide synthase; treadmill running; inotropic effect; chronotropic effect
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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IT IS WELL KNOWN that nitric oxide (NO) is produced in the vascular endothelial cells and has a potent vasodilator effect (34). The heart, as well as the vascular endothelium, produces NO (2, 3, 43, 44). NO is a ubiquitous signaling molecule synthesized in the conversion of L-arginine to L-citrulline by NO synthase (NOS) (28). There are at least three isozymes of NOS: endothelial NOS (eNOS), inducible NOS (iNOS), and neuronal NOS (nNOS) (5, 23, 48). The different cell types comprising the heart express the three isoforms of NOS (2, 3, 43, 44). eNOS is expressed constitutively in cardiac myocytes in rodents and humans (2, 7, 14, 38). iNOS is also expressed in cardiac myocytes, which are expressed in the abnormal heart occurring inflammatory cytokines (3). nNOS is expressed in orthosympathetic nerve terminals conjugating to the myocardium (44) and atrioventricular node (43) but not in cardiac myocytes (37). Also, NO is produced and released by the coronary arterial endothelium in the basal condition. However, it is not known whether the production of NO in the heart is altered by intense exercise.
It has been reported that NO attenuates cardiac myocyte
contraction to 1-adrenergic stimulation (1,
2). It has also been reported that NO inhibits the
positive chronotropic response to 1-adrenergic
stimulation in the cardiac myocyte (1, 18). In the settings of some pathological conditions, such as heart failure,
left ventricular dysfunction, and ischemia, endogenous NO inhibits the
positive inotropic or chronotropic responses to 1-adrenergic stimulation (15,
16, 32). These findings suggest that the
modulation of cardiac function by myocardial NO is altered under some
pathological conditions. However, it is not known whether production of
NO in the heart is altered by physiological stress, e.g., intense
exercise. Intense exercise increases cardiac myocyte contraction, heart
rate, and coronary blood flow. Because NO attenuates positive inotropic
and chronotropic responses to 1-adrenergic stimulation
in the cardiac muscles (1, 2,
18), we hypothesized that the production of NO in the
heart would be changed after an acute bout of exercise.
We investigated whether the production of NO in the heart in
rats is altered by intense exercise. Rats performed treadmill running
for 45 min at a speed of 25 m/min. Immediately after this intense
exercise, the heart was quickly removed and mRNA expression for the
three isoforms of NOS (eNOS, nNOS, and iNOS) and the tissue level of
nitrite (NO2)/nitrate (NO3) (i.e.,
NOx), which are stable end products of NO in the heart, were
determined. We also studied whether the expression of eNOS protein in
the heart is altered by intense exercise.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Animals and protocol. Fifteen male Wistar rats (7 wk old) were obtained from Clea Japan (Tokyo, Japan) and cared for according to the Guiding Principles for the Care and Use of Animals based on the Helsinki Declaration of 1964. The rats were maintained on a 12:12-h light-dark cycle and received food and water ad libitum. All rats were familiarized with running on a motor-driven treadmill 5 days/wk, over a 4-wk period, until they were capable of running 25 m/min for 45 min at no incline (0% grade) according to our previous paper (26). In brief, the running time and the speed of the treadmill were increased gradually over the 4-wk period from 10 min at 10 m/min to 45 min at 25 m/min. Systolic arterial pressure and heart rate of the animals were measured using a tail-cuff sphygmomanometer (model PS-100, Riken Kaihatsu, Kanagawa, Japan) on the day before the experiment. The body weight of the animals was also measured on the day before the experiment. On the day of the experiment the rats were randomly divided into two groups. In one group, eight animals ran on a treadmill (0% grade) for 45 min at a speed of 25 m/min (exercise group). Shepherd and Gollnick (39) reported that this load (25 m/min) of treadmill running in the rat accounts for ~78% of maximal oxygen consumption. The other seven animals remained at rest during this 45-min period (control group).
Immediately after removal from the treadmill, animals in the exercise group were anesthetized with diethyl ether. After the animals were anesthetized, a blood sample was collected from the heart, and the heart was quickly removed, weighed, and frozen in liquid nitrogen. The plasma was stored at
80°C for measurement of plasma norepinephrine
concentration by radioenzymatic assay. The heart samples were also
stored at 80°C for measurement of tissue NOx level, for
determination of the expression levels of mRNAs of three isoforms of
NOS (eNOS, nNOS, and iNOS) by RT-PCR analysis, and for
determination of eNOS protein expression by enhanced chemiluminescence
(ECL) Western blotting analysis. The control animals were
killed ~24 h after their last bout of exercise, i.e., at the same
time point as the exercised rats.
Measurement of plasma norepinephrine concentration. The plasma norepinephrine concentration was measured using a radioenzymatic assay based on the method of Peuler and Johnson (35). Plasma samples from each animal were examined in triplicate.
RT-PCR to determine levels of eNOS mRNA, nNOS mRNA, and iNOS mRNA in the heart. The expressions of eNOS mRNA, nNOS mRNA, and iNOS mRNA in the left ventricle were analyzed by RT-PCR. The expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was determined as an internal control. Semiquantitative RT-PCR was performed according to the method described by Firsov et al. (8) with a minor modification (see below).
Total tissue RNA was isolated by acid guanidinium thiocyanate-phenol-chloroform extraction with Isogen (Nippon Gene, Toyama, Japan) according to the method described in our previous papers (19, 26). Briefly, the tissue was homogenized in Isogen (100 mg tissue/1 ml Isogen) with the Polytron tissue homogenizer (model PT10SK/35, Kinematica, Lucerne, Switzerland). The RNA was extracted with chloroform, precipitated with isopropanol, and washed with 75% (vol/vol) ethanol. The resulting RNA was resolved in diethyl pyrocarbonate-treated water, treated with DNase I (Takara, Shiga, Japan), and extracted again by Isogen to eliminate the genomic DNA. The RNA concentration was determined spectrophotometrically at 260 nm. The RNA (10 µg) was primed with 0.05 µg of oligo d(pT)12-18 and reverse transcribed by avian myeloblastosis virus reverse transcriptase using a first-strand cDNA synthesis kit (Life Sciences). The reaction was performed at 43°C for 60 min. The cDNA was diluted in a 1:10 ratio and used for PCR. Each PCR reaction contained 10 mM Tris · HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, dNTP at 200 µM each, gene-specific primer at 0.5 µM each, and 0.025 U/µl Taq polymerase (Takara). The gene-specific primers were synthesized according to the published cDNA sequences for each of the following: eNOS (21), nNOS (27), iNOS (10), and GAPDH (45). The sequences of the oligonucleotides were as follows: eNOS (sense): 5'-CTGGCAAGACCGATTACACGAC-3'; eNOS (antisense): 5'-GTCCTCACCGCCTTTTCCAG-3'; nNOS (sense): 5'-AATGGAGACCCCCCTGAGAAC-3'; nNOS (antisense): 5'-TCCAGGAGGGTGTCCACCGC-3'; iNOS (sense): 5'-GTACATGGGCACCGAGATTG-3'; iNOS (antisense): 5'-CTACTACTACCAGATCGAGCC-3'; GAPDH (sense): 5'-GCCATCAACGACCCCTTCATTG-3'; GAPDH (antisense): 5'-TGCCAGTGAGCTTCCCGTTC-3'. PCR was carried out using a PCR thermal cycler (model TP-3000, Takara). The cycle profile included denaturation for 15 s at 94°C, annealing for each suitable time at each suitable temperature, and extension for each suitable time at 72°C. The annealing time and temperature were set as follows: 15 s at 66°C for eNOS, 30 s at 62°C for nNOS, 15 s at 65°C for iNOS, and 15 s at 58°C for GAPDH. The extension time was set as follows: 30 s for eNOS and GAPDH and 45 s for nNOS and iNOS. The reaction cycles of PCR were performed in the range that demonstrated a linear positive correlation between the amount of cDNA and the yield of PCR products, i.e., 26 cycles for eNOS, 40 cycles for iNOS, 40 cycles for nNOS, and 20 cycles for GAPDH. The PCR products were found to be of the expected size, as shown by 1.8% agarose gel electrophoresis for eNOS and 1.2% agarose gel electrophoresis for nNOS, iNOS, and GAPDH. In addition, the specificity of the amplified sequences was confirmed by restriction enzyme analysis and DNA sequencing. The DNA sequence of each amplicon was perfectly matched to each published sequence.Semiquantitative analysis of PCR products. The reaction cycle of PCR was performed in the range that demonstrated a linear positive correlation between the amount of cDNA and the yield of PCR products. The amplified PCR products were electrophoresed on 1.8 or 1.2% agarose gels, stained with ethidium bromide, visualized by an ultraviolet transilluminator, and photographed. The photographs were scanned by CanoScan 600 (Canon, Tokyo, Japan), and quantification was performed by computer using MacBAS software (Fuji Film, Tokyo, Japan). The positive-control DNAs for verification of the semiquantitative PCR analysis were prepared from each PCR product derived from rat cDNA. Each PCR product concentration was calculated by assuming that the mass of a nucleotide pair in DNA is 660 Da (36). We performed semiquantitative PCR analysis to evaluate the expression level of eNOS mRNA, nNOS mRNA, iNOS mRNA, and GAPDH mRNA. To demonstrate that our semiquantitative PCR strategy was valid, serial dilutions of each positive-control cDNA were amplified by PCR and quantified by scanner.
Measurement of NOx level in heart.
NOx level in the heart was determined according to the method of Green
et al. (12) with a minor modification. Heart tissues were
homogenized with two volumes of 50 mM Tris · HCl (pH 7.4, 4°C)-0.1 mM EDTA-0.1 mM EGTA-0.5 mM dithiothreitol-1 µM pepstatin A-2 µM leupeptin-1 mM phenylmethylsulfonyl fluoride on ice using a
Teflon homogenizer. The homogenate was centrifuged at 9,000 g for 20 min at 4°C, and the supernatant obtained was
centrifuged at 105,000 g for 60 min at 4°C. The resulting
soluble fraction was stored at 80°C until the NOx assay, and the
pellet (microsomal fraction) was frozen under liquid nitrogen and
stored at 80°C until the eNOS protein assay. For determination of
NOx level, 80 µl of each sample was incubated for 60 min at 25°C in
a 270-µl incubation mixture containing (in µl) 140 of 125 mM KPi
(pH 7.5), 10 of 87.5 µM FAD, 10 of 3.5 mM NADPH, 90 of distilled
water (DW), and 20 nitrate reductase (1.75 U/ml; Sigma, St.
Louis, MO). The reaction was initiated by addition of the nitrate
reductase to convert NO2 to NO3.
The reaction was terminated by addition of 0.8 ml of Griess reagent and
0.45 ml of DW. After each mixture was centrifuged at 14,000 g for 5 min, the supernatants obtained were determined spectrophotometrically at 542 nm.
Electrophoresis and immunoblot analysis for measurement of eNOS
protein in heart.
Heart microsomes were denatured by boiling for 5 min with SDS sample
buffer (62.5 mM Tris · HCl buffer, pH 6.8, containing 25%
glycerol, 2% SDS). Protein concentrations were determined by the
bicinchoninic acid protein assay reagents (Pierce, Rockford, IL) with BSA as a standard (40). The samples were followed
by heat denaturation at 96°C for 5 min with -mercaptoethanol.
Western blot analysis was performed by the method described by North et al. (33) with a minor modification. Briefly, each
microsomal preparation was separated on an SDS-polyacrylamide gel (8%)
and then transferred to polyvinylidene difluoride (Millipore, Tokyo, Japan) membranes at 1 mA/cm2 for 120 min. The membrane was
treated with blocking buffer 5% skim milk in phosphate-buffered saline
contained 0.05% Tween 20 (PBS-T) for 12 h at 4°C. The membrane
was probed with monoclonal anti-eNOS antibody (Transduction
Laboratories, Lexington, KY; 1:2,500 dilution with blocking buffer) for
1 h at room temperature, washed with PBS-T five times, and then
incubated with anti-mouse immunoglobulin antibody, a horseradish
peroxidase-conjugated F (ab')2 fragment from sheep
(Amersham Life Science, Buckinghamshire, UK; 1:2,500 dilution with
blocking buffer) for 1 h at room temperature. After this reaction,
the membrane was washed with PBS-T six times. Finally, the eNOS was
detected by ECL system (Amersham Life Science), and exposed to
Hyper film (Amersham Life Science).
Statistics. Values are expressed as means ± SE. To evaluate differences between the control and the exercise group, Student's t-test for unpaired values was used. P < 0.05 was accepted as significant.
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RESULTS |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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There were no significant differences between the control group
(n = 7) and the exercise group (n = 8)
in systolic arterial pressure [125 ± 3 (n = 7)
vs. 121 ± 3 (n = 8) mmHg] or heart rate [379 ± 16 (n = 7) vs. 387 ± 8 (n = 8) beats/min] on the day before death. There was
no significant difference in body weight between the two groups (Table
1). Neither the left ventricular wet
weight nor the left ventricle weight mass index for body weight
differed significantly between the two groups (Table 1). These results indicated that the physical changes induced by 4 wk of
training, which was performed for familiarization with running on the
treadmill, were of the same degree in the control and the exercise
group.
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Immediately after the 45-min exercise or rest period, the plasma
concentration of norepinephrine was significantly greater in the
exercise group than in the control group (Fig.
1). Thus the plasma concentration of
norepinephrine was greatly increased by this intense exercise.
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To semiquantitatively determine alterations in gene expression of
cardiac NOS isozymes by exercise, the relationship between the amount
of cDNA and the yield of PCR products was examined and the results are
shown in Fig. 2. We used copies as the
unit for the x-axis in Fig. 2, because the use of
copies is considered to be more suitable than the use of micrograms for
showing quantity of PCR for the following reason. The objected
DNA is duplicated every cycle of PCR. Because molecular
weights of DNA fragments are in proportion to the length of DNA, the
weight of each PCR product differs from each in same copy number.
Therefore, we used copies as the unit of x-axis in Fig. 2.
There was a linear correlation between the initial amount of eNOS cDNA
and the yield of PCR products (Fig. 2A). In the cases of
iNOS, nNOS, and GAPDH, the yield of PCR products was also in proportion
to the initial amount of cDNA (Fig. 2, B-D). Under these
conditions, the expression of eNOS mRNA in the heart was markedly and
significantly decreased in the exercise group (Fig.
3A). However, neither the
expression of iNOS mRNA nor nNOS mRNA in the heart differed
significantly between the two groups (Fig. 3, B and
C). These findings suggest that the isozyme-specific
downregulation of NOS mRNA was caused by intense exercise.
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Figure 4 shows the representative film of
immunoblotting for eNOS protein expression in the heart with and
without this intense exercise. The expression level of eNOS protein in
the heart was lower in the exercise group than in the control group
(Fig. 4).
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The NOx level, an index of NO production in vivo, in the heart after
this intense exercise was also significantly lower in the exercise
group than in the control group (Fig. 5).
Therefore, these findings suggest that the production of NO was
significantly decreased in the heart as the result of intense exercise.
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The relationships between the levels of mRNA expressions of three
isozymes of NOS and the level of NOx in the heart are shown in Fig.
6. There was a significant positive
correlation between the level of mRNA expression of eNOS and the level
of NOx in the heart (Fig. 6A). However, there was no
correlation between the levels of mRNAs of nNOS or iNOS and the level
of NOx in the heart (Fig. 6, B and C).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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In the present study, we determined NO production in the heart of
rats after intense exercise. The tissue level of NO2/
NO3 (i.e., NOx) was significantly lower in the
exercised rats than in the control rats. The expression of eNOS mRNA in
the heart was also significantly lower in the exercise rats. However,
neither the expression of iNOS nor nNOS mRNA in the heart differed
significantly between the two groups. There was a significant positive
correlation between the eNOS mRNA and NOx level in the heart, whereas
there was no correlation between the nNOS or iNOS mRNA and NOx level in
the heart. We also demonstrated that expression of eNOS protein in the
heart was decreased by the present intense exercise. Therefore, it is
suggested that the decrease in NOx level in the heart induced by
intense exercise is attributed to a decrease in the expression of eNOS
(both mRNA and protein levels). The present study revealed for the
first time that the production of NO, evaluated by NOx level, in the
rat heart is greatly decreased by intense exercise, suggesting that the
exercise-induced decrease in production of NO in the heart cancels the
NO-mediated attenuation of positive inotropic and chronotropic
responses to 1-adrenergic stimulation, thereby
contributing to the acceleration of increase in myocardial contractility and heart rate during intense exercise.
Cardiac myocytes as well as vascular endothelial cells produce NO, as
evidenced by the expression of NOS mRNA in heart muscles (2, 3, 7, 14,
38). In addition to its vasodilator effect, NO attenuates
the positive inotropic and chronotropic responses to
1-adrenergic stimulation in heart muscles
(1, 2, 14, 18,
22). It has also been reported that the NO produced
through the muscarinic cholinergic receptors pathway causes NOS
activation in the cardiac myocytes and attenuates positive inotropic
and chronotropic responses to 1-adrenergic stimulation (1, 2, 14). Exercise results in
a marked increase in myocardial contractility and heart rate caused
mainly by 1-adrenergic stimulation in the heart. In the
present study, the plasma concentration of norepinephrine was markedly
greater in the exercised rats than in the control rats, suggesting that
sympathetic nerve activity was greatly augmented during acute exercise
under the present conditions. Furthermore, it has been reported that
the present intensity (25 m/min) of treadmill running in rats
prominently enhances heart rate and myocardial contractility
(9). The present study revealed that the production of NO
in the heart is markedly decreased by intense exercise. These findings
suggest that the exercise-induced decrease in production of NO in the
heart cancels the NO-mediated attenuation of positive inotropic and
chronotropic responses to 1-adrenergic stimulation,
thereby contributing to the acceleration of increase in myocardial
contractility and heart rate during intense exercise. Such a decrease
in NO in the heart would contribute to maximize cardiac function
(increase in myocardial contractility and heart rate) during intense exercise.
The mechanism underlying the decrease in the production of NO in the heart by intense exercise remains to be elucidated. The production of NO is considered to be regulated by multiple factors (31). It has been reported that endothelin-1 (ET-1) depresses NOS activity in vascular smooth muscle cells (17). We previously reported that the circulating plasma concentration of ET-1 was significantly increased by intense exercise (25). Cardiac myocytes produce ET-1 (42). We recently reported that the production of ET-1 in the heart is markedly increased by intense exercise in rats (26). The present study showed that the production of NO in the heart is significantly decreased by intense exercise. Therefore, it is possible that, in the heart, increase in myocardial ET-1 by intense exercise contributes to a reduction in the production of NO during intense exercise. During intense exercise, there may be interactions among ET-1, NO, and other factors in the regulation of the production of these substances in the heart. Alternatively, the following mechanism is also possible. Exercise induces hyperemia in the heart. Thus there is a possibility that NOx was washed out from the tissue of heart in the exercised rats and, therefore, that NOx level in the heart was decreased after intense exercise. However, we believe that this possibility is unlikely because we demonstrated that the expression of eNOS (both mRNA and protein levels) in the heart was decreased by intense exercise and that there was a significant positive correlation between the eNOS mRNA and NOx level in the heart. However, the precise mechanism of the decrease in production of NO in the heart by intense exercise remains to be elucidated.
In the heart, cardiac myocytes and vascular endothelial cells produce NO (2, 3). Which cells are the source of the decrease in NO production in the heart by intense exercise is presently unclear. Exercise results in a marked increase in coronary blood flow (11, 13, 20), which in turn might cause the increase in the levels of shear stress on vascular endothelial cells of the coronary vessels. It has been shown that shear stress increases the release of NO in isolated blood vessels and in cultured endothelial cells (31). Furthermore, Bernstein et al. (4) reported that the NOx level in the coronary circulation is increased by exercise. In the present study, the production of NO in the heart, which consists of myocardium and coronary vessels, is markedly decreased by intense exercise. On the basis of the findings of Bernstein et al. (4) and the findings of our present study, it is considered that the decrease in NO production in the heart induced by intense exercise is attributed to the decrease in NO production in the cardiac muscles. However, further study is needed to determine which cells (cardiomyocyte or vascular endothelium) are responsible for this decrease in NO production.
The only known source of endogenous NOx,
NO2/NO3 in mammalian tissues is
considered to be generated through the conversion of
L-arginine to L-citrulline by NOS. NO is
oxidized easily to NO2, which is then converted to
NO3 when it reacts with hemoglobin (30).
In the present study, we examined the tissue levels of NOx in the heart
to estimate NO production after intense exercise. The tissue NOx level
in the heart was significantly lower in the exercise rats than in the
control rats. The change in NOx level would be caused by changes of
activation of eNOS, nNOS, and iNOS. Furthermore, the family of the
three isoforms of NOS (eNOS, nNOS, and iNOS) is expressed in the heart
(2, 3, 38, 43,
44). Therefore, we tried to determine what type of NOS
contributes to the exercise-induced decrease in tissue NOx level in the
heart. In the present study, there was a significant positive
correlation between the eNOS mRNA and NOx level in the heart (see Fig.
6A). However, there was no correlation between nNOS or iNOS
mRNA and NOx level in the heart (see Fig. 6, B and
C). The present study showed that the expression of eNOS
mRNA in the heart is decreased by intense exercise. We also
demonstrated that expression of eNOS protein in the heart was decreased
by intense exercise. It was reported that the change of NOx level
corresponded to that of eNOS expression (both mRNA and protein levels)
in rats with chronic renal insufficiency (46). Taken
together, it is considered that the decrease in NOx level in the heart
induced by intense exercise is attributed to a decrease in the
expression of eNOS (both mRNA and protein levels). Because eNOS-derived
NO attenuates the positive inotropic and chronotropic responses to
1-adrenergic stimulation in heart muscles
(1, 2, 15), it was considered
that the decrease in NO production in the heart induced by intense
exercise causes the cancellation of the NO-mediated attenuation of
positive inotropic and chronotropic responses to
1-adrenergic stimulation, thereby contributing to the
acceleration of increase in myocardial contractility and heart rate
during intense exercise.
In summary, we demonstrated that the tissue NOx level and the
expression of eNOS mRNA in the heart were significantly lower in the
exercised rats than in the control rats. There was a significant positive correlation between the eNOS mRNA and NOx level in the heart.
The present study also demonstrated that the expression of eNOS protein
in the heart was decreased by intense exercise. Therefore, it is
suggested that the decrease in NOx level in the heart induced by
intense exercise is attributed to a decrease in the expression of eNOS
(both mRNA and protein levels). The present data reveal for the first
time that the production of NO in the heart is markedly decreased by
intense exercise. Therefore, the present study provides the possibility
that the exercise-induced decrease in production of NO in the heart
cancels the NO-mediated attenuation of positive inotropic and
chronotropic responses to 1-adrenergic stimulation,
thereby contributing to the acceleration of increase in myocardial
contractility and heart rate during intense exercise. Therefore, it is
possible that myocardial NO contributes to the modulation of cardiac
function during intense exercise.
Perspectives
The present study demonstrated for the first time that the production of NO in the heart is markedly decreased by intense exercise and therefore provided a new hypothesis that exercise-induced increase in myocardial contractility is partly attributed to the cancellation of the NO-mediated attenuation of positive inotropic response to1-adrenergic stimulation. To support this hypothesis, further study is needed to determine whether a decrease in NO production by intense exercise causes a change in cardiac function in
vivo. A study examining whether specific inhibition of cardiac NO
production affects cardiac function in vivo would provide an important
finding for this hypothesis.
NG-nitro-L-arginine methyl ester
(L-NAME) and
NG-monomethyl-L-arginine acetate
(L-NMMA) are widely used as inhibitors for NO production;
however, L-NAME or L-NMMA inhibits NO
production by both vascular endothelial cells and cardiomyocytes
(29). Application of L-NAME or
L-NMMA in vivo causes a marked elevation of blood pressure
(29), which makes the assessment of whether changes of
myocardial function by L-NAME or L-NMMA are
attributed to direct inhibition of myocardial NO production or to
change of afterload to the heart difficult. Suto et al.
(41) reported that application of L-NMMA in
vivo in dogs produces an increase in the slope of the end-systolic
pressure-volume relationship, an indicator of myocardial
contractility in vivo, as well as an increase in systemic blood
pressure. This report supports a hypothesis that endogenously
generated NO in cardiomyocytes is involved in myocardial function in
vivo, although this report has a study limitation that change in
myocardial function by L-NMMA in vivo is caused by change
of afterload to the heart, i.e., change of systemic blood pressure.
Currently, there is no available compound that specifically inhibits
myocardial production of NO in vivo. A development of such a
compound is expected, because it is important to study whether
myocardial function during intense exercise is affected by the presence
of a specific inhibitor for myocardial NO production in vivo.
NO has potent vasodilator effects in the coronary vessels
(34) and attenuates positive inotropic and chronotropic
responses to 1-adrenergic stimulation in the cardiac
muscles (1, 2, 15). Therefore,
it has been considered that NO has cell-specific effects in the heart.
Furthermore, NO has been shown to be involved in angiogenesis
(24, 49). It has been reported that the NOx level in the coronary circulation is increased by exercise
(4). Although the present study showed that the production
of NO in the heart, which consists of myocardium and coronary vessels, was markedly decreased by intense exercise, the production of NO in the
coronary vessels may be increased by intense exercise. The repeated
bouts of exercise induced angiogenesis in the heart (6,
47). Therefore, it is of great interest to determine whether the NO system in the heart contributes to the angiogenic response to exercise.
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ACKNOWLEDGEMENTS |
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This study was supported by grants-in-aid for scientific research from the Ministry of Education, Science, Sports, and Culture of Japan (00006781, 10670629, 11480003, 11557047), by a grant from University of Tsukuba Research Projects, and by a grant from the Miyauchi Project of Center for Tsukuba Advanced Research Alliance in University of Tsukuba.
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FOOTNOTES |
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Address for reprint requests and other correspondence: T. Miyauchi, Cardiovascular Division, Dept. of Internal Medicine, Institute of Clinical Medicine, Univ. of Tsukuba, Tsukuba, Ibaraki 305-8575, Japan (E-mail: t-miyauc{at}md.tsukuba.ac.jp).
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. §1734 solely to indicate this fact.
Received 3 January 2000; accepted in final form 12 April 2000.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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; n = 8) and the control rats
(
; n = 7). The value of the level of
expression of eNOS mRNA, iNOS mRNA, and nNOS mRNA was normalized by
that of GAPDH mRNA.