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ABSTRACT |
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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The current study was designed to test the hypothesis that endurance training improves the ability of the diaphragm muscle to resist exercise-induced oxidative stress. Twenty-eight male Wistar rats were assigned to either untrained or trained groups. Trained rats were treadmill-trained for 9 wk. Each group was subdivided into acutely exercised or nonexercised groups. Diaphragm muscle from each rat was analyzed to determine the levels of certain antioxidant enzymes: Mn-superoxide dismutase (Mn-SOD), Cu,Zn-superoxide dismutase (Cu,Zn-SOD), glutathione peroxidase, and catalase. In addition, interleukin-1 and myeloperoxidase levels were determined. Endurance training upregulated all of the antioxidant enzymes. Conversely, acute exercise increased glutathione peroxidase and catalase in untrained rats, while it had no overt effect on any antioxidant enzymes in trained rats. Both Mn-SOD and Cu,Zn-SOD contents and activities were increased with endurance training. However, the mRNA expressions of both forms of SOD did not show any significant change with endurance training. Acute exercise also increased the levels of interleukin-1 and myeloperoxidase in untrained rats but not in trained rats. Moreover, acute exercise significantly increased the ability of neutrophils to produce superoxide, especially in untrained rats. The results from this study demonstrate that endurance training can upregulate certain antioxidant enzyme activities in rat diaphragm muscle, indicating the potential for improvement of the resistance to intracellular reactive oxygen species. The results of this study also suggest that acute exercise may cause oxidative damage in rat diaphragm through the activation of the inflammatory pathway and that endurance training may minimize such an extracellular oxidative stress by acute exercise.
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INTRODUCTION |
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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The diaphragm is a unique skeletal muscle that works continuously, unlike other skeletal muscles. Thus, the diaphragm may always be exposed to some degree of oxidative stress. The oxygen uptake of active muscles (such as the diaphragm) markedly increases during exercise, leading to an increased generation of reactive oxygen species (1). The possibility has been proposed that the reactive oxygen species may cause exercise-induced oxidative damage in active muscles (2, 3). Davies and associates (2), for example, have reported that the levels of reactive oxygen species in muscle doubles after exhaustive exercise. Reactive oxygen species are well known to be able to cause a wide spectrum of cellular damage due to enzyme inactivation, lipid peroxidation, nucleic acid damage, and the like (3). Recently, there is growing evidence that free radical-induced injury may occur in respiratory muscles during periods of oxidative stress, probably leading to clinically relevant forms of respiratory muscle dysfunction or fatigue (4). On the other hand, mammalian tissues contain enzymatic and nonenzymatic antioxidant defense systems that protect or minimize oxidative tissue damage caused by reactive oxygen species. Current literature indicates that exercise training can enhance both antioxidant capacity and oxidative capacity in skeletal muscle, including the diaphragm muscle (7). However, few studies utilizing the diaphragm muscle have been documented (11, 12). Although Powers and coworkers (11, 12) demonstrated that the activities of superoxide dismutase (SOD) and/ or glutathione peroxidase (GPX) in costal diaphragm were upregulated by endurance training, there was no data about catalase (CAT) or SOD isoenzymes. Therefore, there appears to still exist a paucity of data concerning the effect of exercise training on the antioxidant enzyme system in the diaphragm muscle. In addition, to our knowledge, there is no data available about the effect of acute exercise on the antioxidant enzyme system in trained diaphragm.
The specific aim of the current study was to test the hypothesis that endurance training improves the ability of the diaphragm muscle to resist exercise-induced intracellular and
extracellular oxidative stress, focusing on the alterations of antioxidant enzymes and of inflammatory mediators such as interleukin-1 (IL-1) and neutrophils. SOD, which serves as a
scavenger of superoxide (O
2 .), is one of the most important
enzymes in the antioxidant defense system. Mammalian tissues contain two forms of SOD: Mn-SOD is present mostly
in the mitochondrial matrix, whereas Cu,Zn-SOD is predominantly localized in the cytosol. Therefore, the effects of endurance training and acute exercise on the antioxidant enzyme system in the rat diaphragm muscle were investigated. To estimate the effect of exercise on the antioxidant enzyme system,
the activities of two other major antioxidant enzymes, GPX
and CAT, were also measured. On the other hand, neutrophils
are considered to be one of the main sources of extracellular
reactive oxygen species. We thus measured myeloperoxidase
(MPO) (found in abundance in neutrophils) in the diaphragm
muscle and the ability of neutrophils to generate O2 . in response to exercise as an index of extracellular oxidative stress.
Strenuous exercise is well known to induce inflammatory
cytokines such as IL-1 (14). Consequently, IL-1 may cause oxidative tissue damage because of the increased generation of
reactive oxygen species by activated neutrophils. IL-1 has also
been reported to induce Mn-SOD (15, 16). Therefore, IL-1
seems to activate both of oxidant and antioxidant pathways,
and strenuous exercise may influence the antioxidant enzyme
system via inflammatory mediators. On the other hand, Sprenger
and colleagues (17) demonstrated no change in plasma IL-1
when well-trained runners completed a 20-km road race. So,
we hypothesize that endurance training not only improves the
antioxidant capacity of tissues for intracellular oxidative stress
but also decreases the generation of exercise-induced extracellular oxidative stress through inflammatory process.
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METHODS |
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INTRODUCTION
METHODS
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DISCUSSION
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Animal Preparation
Twenty-eight male Wistar rats weighing 90 to 110 g, at 5 wk of age, were obtained from Japan SLC (Shizuoka, Japan). Rats were caged in pairs and placed at 24° C under artificial lighting for 12 h from 7 A.M. to 7:00 P.M. Rats had free access to food and water. The animals were cared for in accordance with the Guiding Principles for the Care and Use of Animals approved by the Council of the Physiological Society of Japan based upon the Helsinki Declaration, 1964.
Seven-week-old rats were divided randomly into two groups: untrained (U, n = 14) and trained (T, n = 14) groups. The T group was exercised on a rodent treadmill, set at an 8° incline, 5 d/wk for 9 wk. During the first week, rats were acclimated to the treadmill. The initial training intensity was 15 m/min for 20 min; thereafter, the running speed and duration were progressively increased until, after 6 to 7 wk, the rats ran continuously for 90 min/d, 30 m/min. Over the equivalent 9-wk period, the U group was sedentary except for the final week, when this group was also acclimated to treadmill running (15 to 20 m/min, 10 min/d), every 3 d, and three times in total). Both U and T groups were then subdivided into rats to be killed at rest (UR and TR) and rats to be killed immediately after an acute bout of exercise (UE and TE). About 48 h after the last acclimation running or 48 h after the last training session, an acute bout of exercise in the UE or TE group was performed. The intensity of acute exercise performed by the UE and TE rats was 20 m/min for 90 min and 0° incline and 30 m/min for 90 min and 8° incline, respectively. To eliminate diurnal effects, all animals were killed at the same time (9:00 A.M. to 12:00 P.M.) and at the same age (4 mo old).
Assay for Antioxidant and Oxidative Enzyme Activities
After rats were killed by decapitation and exsanguinated, costal diaphragms were quickly excised and frozen in liquid nitrogen. The muscle tissues were stored at 
80° C for later analysis. Subsequently, a
portion of muscle tissues was thawed in an ice-cold medium containing 0.25 M sucrose, 10 mM 2-amino-2-hydroxymethyl-1,3-propanediol (Tris), and 0.1 mM EDTA (pH 7.4, wt/vol 1:9), minced, and homogenized on ice in brief bursts by a Polytron homogenizer (Kinematika, Luzern). The homogenate was centrifuged at 750 × g (4° C) for 15 min, and the supernatant was used for various assays.
Total SOD (ED 1.15.1.1) activity was determined using the method of Crapo and associates (18). One unit of SOD activity was defined as the amount required to inhibit the rate of reduction of cytochrome c by 50%. Mn-SOD was distinguished from Cu,Zn-SOD according to the cyanide procedure containing potassium cyanide at 1 mM final concentration in the reaction mixture. GPX (EC 1.11.1.9) activity was assayed spectrophotometrically according to Tappel (19). CAT (EC 1.11.1.6) activity was measured by the method of Aebi (20). Citrate synthase (EC 4.1.3.7) activity as an index of physical training was determined according to Shepherd and Garland (21). Protein content was determined by the method of Lowry and coworkers (22).
ELISA for Immunoreactive Mn-SOD and Cu,Zn-SOD Contents
Rat Mn-SOD was purified from livers of male Wistar rats. A polyclonal antibody against the purified rat Mn-SOD was raised in rabbits and purified by precipitation with 50% saturated ammonium sulfate, DEAE-cellulose chromatography, and immuno-affinity column chromatography using purified Mn-SOD as an absorbent. The specificity of this antibody was judged by Western blotting analysis. An ELISA was developed with the polyclonal antibody using a sandwich method. The biotinylated antibody was prepared by incubating the mixture of the antibody and N-hydroxysuccinimidobiotin and used as the secondary antibody. An ELISA for Cu,Zn-SOD was also established in the same manner as for Mn-SOD. Immunoreactive Mn-SOD and Cu,Zn-SOD contents were measured by the ELISA methods as previously described (23). Polystyrene microtiter plates were coated with the antibody (2 µg/ml) and incubated at 4° C overnight. Plates were washed three times with buffer A (10 mM phosphate-buffered saline [PBS], pH 7.4, containing 0.05% Tween 20) and then incubated for 2 h at 25° C with buffer B (0.1% bovine serum albumin in PBS, pH 7.4) to block the free binding sites. Samples and standard solutions (purified rat Mn-SOD or Cu,Zn-SOD) were diluted with buffer B and then 100-µl aliquots of the diluted samples and standards were added to the antibody-coated plates. After incubation for 2 h at 25° C, the plates were washed with buffer A three times. Then, 50-µl aliquots of the secondary antibody were added to each well. After incubation for 2 h at 25° C, the plates were washed with buffer A three times, and 50-µl aliquots of horseradish peroxidase-conjugated streptavidin (Dako Japan Co., Tokyo, Japan) solution (diluted 1:5,000 with PBS) were added to each well. After incubation for 15 min at 25° C, unbound avidin D solution was removed. The substrate for horseradish peroxidase was then added to the wells (50 µl of 0.003% H2O2 in 0.1 M sodium citrate buffer, pH 5.0, containing 0.6 mg o-phenylenediamine/ml). The color was developed for 5 to 10 min at 25° C and stopped by addition of 1 N sulfuric acid. Absorbance was read at 492 nm using a Titertek Multiskan MCC340 microplate reader (Labsystems, Helsinki, Finland). A calibration curve was prepared from serial 1:2 dilutions of standards (purified Mn-SOD or Cu,Zn-SOD), and the calibration curves for Mn-SOD and Cu,Zn-SOD were linear from 5 to 100 ng/ml and from 0.1 to 2.0 ng/ml, respectively.
Expressions of Mn-SOD and Cu,Zn-SOD mRNA
Total RNA from samples was isolated by the acidic guanidinium
isothiocyanate method of Chomczynski and Sacchi (24). RNA (20 µg/
lane) was separated by electrophoresis on a 1.5% agarose/formaldehyde gel and transferred onto a nitrocellulose filter in 20× SSC (1× SSC = 150 mM sodium chloride/15 mM sodium citrate, pH 7.0) (25). The filter was hybridized to the cDNA probe (which was labeled with
[32P]dCTP by the random-priming method of Feinberg and Vogelstein [26]) encoding rat Mn-SOD protein or rat Cu,Zn-SOD protein
as previously described by our group (27). These probes were obtained by Eco RI and Bam HI digestion of pUC 199 containing the sequences of rat Mn-SOD or rat Cu,Zn-SOD in its Sma I restriction site. Hybridization was performed in a buffer containing 40% formamide, 4× SSC, 1× Denhardt's solution, 10% dextran sulfate, 0.1% sodium dodecyl sulfate (SDS), 40 µg/ml salmon sperm DNA, and 20 mM Tris HCl (pH 7.5) at 42° C for 18 h. The filter was washed in 2× SSC/0.1% SDS at 25° C for 20 min twice and in 1× SSC/0.1% SDS at 50° C for 20 min and then exposed to Amersham Hyperfilm at 70° C. Autoradiographic signals were quantitated using an LKB Ultroscan XL Enhanced Laser Densitometer. To ascertain the integrity and amount of
the RNA sample, 5 µg of each sample was electrophoresed, and the
density of the 18S (2.1 kb) and 28S (5.1 kb) ribosomal RNAs was
quantitated by scanning the negatives of the prints of the ethidium
bromide-stained gel. The degree of SOD isoenzyme mRNA was calculated after normalization to the intensity of the 18S and 28S rRNAs
(an internal control) (28).
Thymocyte Proliferation Assay
Thymocyte proliferation activity was determined as an index of the biologic activity of IL-1 according to the method of Mizel (29), with slight modifications. Briefly, 1.5 × 106 murine (C3H/HeJ) thymocytes in 0.2 ml of complete RPMI 1640 were added in 96-well plates with phytohemagglutinin (Difco) (16 ng/ml) and the supernatants for 72 h. [3H]thymidine (0.5 µCi/well) was added for the final 6 h of culture. Cells were then collected on glass fiber strips by using an automatic cell harvester. Radioactivity (counts per minute [cpm]) was determined by using a liquid scintillation counter. Results are expressed as [3H]thymidine incorporation by cells.
MPO Assay
MPO (EC 1.11.1.7) activity was assayed spectrophotometrically as previously described by Warren and associates (30). Briefly, a portion of diaphragm muscle tissue was homogenized with a Polytron homogenizer, using the homogenization buffer (50 mM phosphate, pH 6.0) containing 0.5% hexadecyltrimethyl ammonium bromide (Wako, Tokyo, Japan) and 5 mM EDTA. The homogenized sample was then sonicated and centrifuged (3,000 × g; 30 min) at 4° C. MPO activity in the supernatant was determined by measuring the change in A460 resulting from decomposition of H2O2 in the presence of o-dianisidine. The value of MPO activity was expressed as A460/min/g tissue.
Assay for the Ability of Neutrophil to Generate Superoxide
The ability of neutrophil to generate O2 . was determined as the intensity of 2-methyl-6( p-methoxyphenyl)-3,7-dihydroimidazo[1,2-a]pyrazine-3-one (MCLA; Tokyo, Kasei, Tokyo)-dependent chemiluminescence according to the method of Nakano and coworkers (31), with a
slight modification. Briefly, rats were anesthetized with enflurane, and
blood was drawn into a heparinized syringe from a tail vein. Leukocytes were isolated by sedimentation in the presence of dextran followed by brief hypotonic lysis of contaminating erythrocytes. The resultant leukocytes, which contained 80 to 95% granulocytes, were
suspended to 1 × 107 cells/ml in Hank's balanced salt solution (HBSS)
and were kept at 0° C for no longer than 2 h prior to use. Zymosan
(Sigma Chemical Co., St. Louis, MO) was opsonized in rat serum at
37° C for 30 min, and opsonized zymosan (OZ) was stored at 80° C
until needed. MCLA was dissolved in distilled water and stored at
80° C until needed. Reaction mixture contained 1 × 105 granulocytes, 2.0 mg of OZ, and 1 µM of MCLA in 2.0 ml of continuously stirred HBSS. The intensity of luminescence was monitored with the
luminescence reader BLR-301 (Aloka Co., Tokyo, Japan) set at 37° C. The reactions were started by the addition of MCLA + OZ; Cu,Zn-SOD (Wako) was added at the end of the reaction; and the maximum
intensity of OZ-stimulated granulocytes was measured as a peak
value. The reaction without OZ was monitored as a control value, and
the difference between a peak value and a control value was regarded
as the ability of granulocytes to generate O2 .. The great majority of
granulocytes were neutrophils, and the chemiluminescence obtained
in the current study was, therefore, considered to be derived from
neutrophils. The data were expressed as percentages of the UR group.
Statistical Analysis
Data are expressed as mean ± SEM. The statistical significance of the data was assessed by a two-way (endurance training × acute exercise) analysis of variance (ANOVA) and the Bonferroni post hoc comparison. When applicable, the unpaired Student's t test was used. A 0.05 level of significance was used.
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Citrate Synthase (CS) Activity
To assess the effect of the endurance training, CS activity was measured in costal diaphragm. CS activity in T rats was significantly higher than in U rats (TR = 0.350 ± 0.005 U/mg protein, UR = 0.250 ± 0.031 U/mg protein; p < 0.05). The result of CS activity indicates that the treadmill-training program used was sufficient to increase oxidative capacity in diaphragm.
Mn-SOD and Cu,Zn-SOD Activities
As shown in Figure 1A, Mn-SOD activity in T rats was higher than in U rats both at rest (51%) and after acute exercise (43%). On the other hand, acute exercise did not influence Mn-SOD activity in either T or U rats. Cu,Zn-SOD activity in T rats was also significantly increased both at rest (25%) and after acute exercise (36%) (Figure 1B), and acute exercise had no effect in either T or U rats. The degree of the training-induced increase in Mn-SOD activity was not statistically different from that in Cu,Zn-SOD activity.
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significantly different from UE: p < 0.05.
Immunoreactive Mn-SOD and Cu,Zn-SOD Contents
As shown in Figure 2A, the content of immunoreactive Mn-SOD in diaphragm was significantly increased with endurance training (22%, TR versus UR), but Mn-SOD content was not affected by acute exercise. Immunoreactive content of Cu,Zn-SOD also showed a training-induced increase (37%, TR versus UR) but no acute exercise-induced increase (Figure 2B). Consequently, neither endurance training nor acute exercise influenced the specific activity of either SOD isoenzyme (Figures 3A and 3B). Therefore, the training-induced increase in both SOD isoenzyme activities is considered to be due to an increase of the enzyme protein.
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Effects of Exercise on Mn-SOD and Cu,Zn-SOD mRNA Expressions
A representative Northern blot analysis for Mn-SOD mRNA and Cu,Zn-SOD mRNA is shown in Figure 4. We could see three major bands (1, 2.4, and 4 kb) of Mn-SOD mRNA in the autoradiogram, as previously reported by Kayanoki and colleagues (32), and those three bands were quantitated. As for Cu,Zn-SOD, a single band (0.7 kb) was detected in the autoradiogram. Unlike the activities and contents of SOD isoenzymes, neither endurance training nor acute exercise statistically affected the expression of Mn-SOD or Cu,Zn-SOD mRNA (Figure 5). Therefore, the levels of SOD isoenzymes appeared to be regulated by translational and/or post-translational mechanism(s).
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GPX and CAT Activities
GPX activity (Figure 6A) in TR rats was significantly greater than that in UR rats (27%), while there was no significant difference between UE and TE rats. Acute exercise increased GPX activity in U rats but not in T rats. CAT activity (Figure 6B) in T rats was significantly higher than that in U rats (77%, TR versus UR; 29%, TE versus UE). Acute exercise enhanced CAT activity only in U rats (p < 0.05, UR versus UE). However, the level of CAT activity in UE rats was less than in TE rats.
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Thymocyte Proliferation Activity
As shown in Figure 7, endurance training did not influence thymocyte proliferation activity as an index of IL-1 activity in rat diaphragm (UR versus TR). However, acute exercise markedly increased thymocyte proliferation activity in U rats (262%) but not in T rats.
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MPO Activity
MPO activity was measured as an index of the influx of neutrophils in rat diaphragm (Figure 8). Endurance training did not alter the level of MPO activity, but acute exercise approximately doubled the level of MPO activity in U rats.
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Ability of Neutrophils to Generate Superoxide
Since the effect of sodium azide, a scavenger of singlet oxygen,
was negligible according to Nakano and coworkers (31), the
chemiluminescence measured was considered to be mostly derived from O2 . production. The effects of endurance training
and acute exercise on the ability of neutrophils to generate O 2 .
are shown in Figure 9. No difference in the ability of neutrophils to generate O 2 . between T and U rats at rest was evident.
On the other hand, acute exercise significantly increased the
ability of neutrophils to generate O 2 . in both groups. In addition, the increase of the activity in U rats was markedly higher
than in T rats (336% in U rats versus 75% in T rats).
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Antioxidant Enzyme Activity
SOD, GPX, and CAT are regarded as the first line of the antioxidant defense system against oxygen free radicals generated during strenuous exercise. The current study showed that endurance training enhanced the activities of all the antioxidant enzymes examined (Mn-SOD, Cu,Zn-SOD, GPX, and CAT) to greater or lesser degree. In addition, acute exercise enhanced GPX and CAT activities in the diaphragm of untrained rats, although it did not increase SOD activity. Acute exercise did not, however, influence any of these enzyme activities in trained rats.
Growing evidence suggests that endurance training upregulates antioxidant enzymes in tissues actively involved in exercise, although controversy still remains. Several investigators
(7, 8, 10, 11) have shown that total (Mn-SOD + Cu,Zn-SOD)
or Mn-SOD activity in skeletal muscle, including diaphragm,
increases significantly after training. In addition, there is evidence that the SOD isoenzyme that can be induced by oxidative stress is Mn-SOD (8, 33). For example, Higuchi and colleagues (8) have reported an increased activity in Mn-SOD
but no change of Cu,Zn-SOD activity with endurance training
in the soleus muscle. The results of the current study, however, showed a training effect on both Mn-SOD and Cu,Zn-SOD. In mitochondria, O2 . is readily converted to H 2O2 by
Mn-SOD, whereas, in cytosol, Cu,Zn-SOD is responsible for
the removal of O2 .. The difference between the results of
Higuchi and colleagues and those of this study may be due to
the difference of muscles used, the animal's sex, and/or assay
methods as well as training programs.
With respect to GPX, most studies, including the current
study, show an increase in GPX activity in skeletal muscle after training (9). Most studies have reported no change in
muscle CAT with training, although Jenkins and coworkers
(7) have reported a significant correlation between CAT activity and 
O2 max in human skeletal muscle. The effect of
training on CAT activity in diaphragm, however, has not been
reported yet. The results of this study indicate an upregulation
of CAT activity with endurance training in diaphragm.
GPX and CAT are the major enzymes that eliminate H2O2 in cells. Since GPX is mostly located in mitochondria and cytosol and CAT exists mainly in peroxisomes, each of them may play a major role in eliminating H2O2. The upregulation of Mn-SOD, Cu,Zn-SOD, and GPX by endurance training is thought to be one of the adaptation phenomena to efficiently eliminate reactive oxygen species produced during physical exercise and minimize damage caused by reactive oxygen species.
Only one study has been published regarding the effect of acute exercise on antioxidant enzymes in diaphragm. Lawler and associates (34) reported no change in SOD or GPX activities in response to acute exercise. According to some researchers, however, acute exercise can enhance antioxidant enzyme activity in limb skeletal muscle (13, 35, 36). In the current study, acute exercise increased the activities of GPX and CAT in diaphragm of untrained rats but not the activities of Mn-SOD Cu,Zn-SOD. These results seem to be in accordance with the work of Ji and coworkers in limb skeletal muscle (36). However, Ji (13) also has reported that exhaustive exercise increases the activity of SOD as well as GPX and CAT in limb skeletal muscle, and SOD may also be inducible by strenuous exercise, although acute exercise used in the current study failed to increase SOD activity in diaphragm.
Immunoreactive Mn-SOD and Cu,Zn-SOD Content
Until now, enzymatic assays have been generally utilized for the determination of SOD level. Compared with enzymatic methods, immunochemical assays for SOD appear to be more reliable because the determinations are specific to the protein moiety (37). Therefore, not only an enzymatic assay but also an immunochemical assay for the determination of SOD level was used in this study. Both immunoreactive Mn-SOD and Cu,Zn-SOD contents were upregulated by endurance training and the increased activities in both SOD isoenzymes seem to be due, mainly, to the increases of the enzyme content.
This study did not show an upregulation of either Mn-SOD or Cu,Zn-SOD mRNA expression with exercise training or acute exercise. Therefore, the increase in SODs during training appears to not be controlled by a transcriptional process but by some post-transcriptional mechanisms, i.e., by a translational and/or a post-translational process. In consideration of the upregulation of immunoreactive SOD contents to endurance training, it may also be possible that the expressions of SOD mRNA were initially upregulated and then downregulated. The precise mechanism, however, must await further study.
Plasma IL-1 level is known to be increased by strenuous exercise (14) and Mn-SOD is induced by IL-1 (15, 16). IL-1 can seem to induce both antioxidant and oxidant pathways. On
the other hand, Masuda and associates (15) have suggested
that the induction of Mn-SOD by IL-1 may be mediated
through the induction of intracellular superoxide by IL-1. Furthermore, Kayanoki and coworkers (32) have indicated that
IL-1 induces the expression of Mn-SOD mRNA and the generation of intracellular peroxides. Therefore, it may be possible that IL-1 stimulation is an oxidant stress, although the precise mechanism for the induction of Mn-SOD by IL-1 still remains unclear. The results obtained from the thymocyte
proliferation assay may suggest that the level of IL-1 in diaphragm of untrained, but not trained, rats increases in response to acute exercise. Sprenger and colleagues (17) found
no change in plasma IL-1 when well-trained runners completed a 20-km road race. Therefore, the decreased response
of thymocyte proliferation activity to acute exercise in trained
rats seems to be one of the adaptative phenomena to endurance training. This study failed to show an upregulation of the
expression of Mn-SOD mRNA despite the increased level of
IL-1 to acute exercise in untrained diaphragm. However, immunoreactive SOD contents were significantly upregulated
with endurance training. On the other hand, the activities of
GPX and CAT in untrained diaphragm increased in response
to acute exercise, and there was a positive correlation between
IL-1 level and GPX (r = 0.808, p < 0.05) and between IL-1
level and CAT activity (r = 0.790, p < 0.05) only in the U
group. Previous study (38) has demonstrated that the administration of IL-1
increases the activities of antioxidant enzymes
in heart (including GPX and CAT). Therefore, our results may
suggest that acute exercise upregulates the level of antioxidant
enzymes through inflammatory cytokine(s) including IL-1 and
that endurance training tends to inhibit such an inflammatory
response to acute exercise.
Since one of the main functions of IL-1 is to activate neutrophils, tissue oxidative damage is expected to be caused by
activated neutrophils during strenuous exercise. The neutrophilic enzyme, MPO, has been shown to provide an excellent
quantitative index of neutrophils in tissues, and its activity has
been reported to be elevated with prolonged running in most
rat tissues, including skeletal muscle (39). Histologic analysis
by Reid and coworkers (40) has shown that inflammatory cells
were attracted to costal diaphragm during resistive breathing.
The current study also showed that MPO activity in diaphragm
of untrained rats increased 2-fold after acute exercise, probably indicating the increased influx of neutrophils into the diaphragm during exercise. Furthermore, the ability of circulating
neutrophil to generate O2 . markedly increased in untrained
rats after acute exercise; in contrast, only a mild, though significant, increase was observed in trained rats. Strenuous exercise is likely not only to increase the influx of neutrophils in
the diaphragm of untrained rats but also to enhance the ability
of neutrophils to produce O 2 .. Therefore, in comparison with
trained diaphragm, untrained diaphragm is exposed to more
oxidative stress not only by intracellular reactive oxygen species but also by extracellular reactive oxygen species. This
study implies that the increased influx of neutrophils into the
diaphragm and this activation may be induced, in part, through
IL-1 and that endurance training may minimize tissue damage by extracellular reactive oxygen species as well as intracellular reactive oxygen species.
In summary, this study has demonstrated that endurance training improves the antioxidant enzyme system in rat diaphragm. Further, strenuous exercise may cause oxidative damage in the diaphragm through the activation of an inflammatory pathway, including IL-1 production and neutrophil activation, whereas endurance training reduces such a response to strenuous exercise. Therefore, the results indicate that the susceptibility to both intracellular and extracellular oxidative stress in rat diaphragm during exercise is improved by endurance training.
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Footnotes |
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Correspondence and requests for reprints should be addressed to Hideki Ohno, M.D., Ph.D., National Defense Medical College, 3-2, Namiki, Tokorozawa 359, Japan.
(Received in original form November 11, 1996 and in revised form June 26, 1997).
Acknowledgments: The writers thank Prof. John O. Holloszy (Washington University, St. Louis, MO) for useful comments on the manuscript, Dr. Keiichiro Suzuki and Prof. Naoyuki Taniguchi (Osaka University, Osaka, Japan) for providing us with polyclonal antibodies for rat Mn-SOD and Cu,Zn-SOD, and Masahiko Segawa (National Defense Medical College, Tokorozawa, Japan) for excellent technical help.
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References |
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DISCUSSION
REFERENCES
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