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Pulmonary Division, Department of Medicine, Case Western Reserve University and MetroHealth Medical Center, Cleveland, Ohio 44109
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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The purpose of the present study was to determine whether it is possible to alter the development of fatigue and ablate free radical-mediated lipid peroxidation of the diaphragm during loaded breathing by administering oxypurinol, a xanthine oxidase inhibitor. We studied 1) room-air-breathing decerebrate, unanesthetized rats given either saline or oxypurinol (50 mg/kg) and loaded with a large inspiratory resistance until airway pressure had fallen by 50% and 2) unloaded saline- and oxypurinol-treated room-air-breathing control animals. Additional sets of studies were performed with animals breathing 100% oxygen. Animals were killed at the conclusion of loading, and diaphragmatic samples were obtained for determination of thiobarbituric acid-reactive substances and assessment of in vitro force generation. We found that loading of saline-treated animals resulted in significant diaphragmatic fatigue and thiobarbituric acid-reactive substances formation (P < 0.01). Oxypurinol administration, however, failed to increase load trial time, reduce fatigue development, or prevent lipid peroxidation in either room-air-breathing or oxygen-breathing animals. These data suggest that xanthine oxidase-dependent pathways do not generate physiologically significant levels of free radicals during the type of inspiratory resistive loading examined in this study.
free radicals; skeletal muscle; diaphragm; respiratory muscles
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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RECENT WORK has shown that oxygen-derived free radicals are generated in the respiratory muscles in response to strenuous contractions (4, 15, 16). Moreover, several experiments have suggested that the free radicals so produced may play a role in inducing respiratory muscle fatigue (1, 12, 18, 24, 26, 28).
The source of these free radicals and the specific intracellular sites altered by reaction with these free radicals remain, however, unclear. In theory, several metabolic pathways could contribute to free radical generation during contraction, including superoxide production by the mitochondrial electron transport chain and the xanthine oxidase-mediated degradation of hypoxanthine (3, 7). Of note, one recent report examining the effect of selenium depletion (selenium is an essential cofactor for xanthine oxidase activity) supports the possibility that xanthine oxidase pathways may play a central role in the generation of free radicals by the loaded diaphragm (1).
If xanthine oxidase-mediated free radical generation is an important source of free radicals and free radical-mediated muscle dysfunction during respiratory loading, then administration of a xanthine oxidase inhibitor should reduce the degree of respiratory muscle fatigue induced by loaded breathing. The purpose of the present study was, therefore, to determine whether it is possible to improve respiratory system performance, alter the development of fatigue, and/or ablate free radical-mediated lipid peroxidation of the diaphragm during loaded breathing by administering oxypurinol, a xanthine oxidase inhibitor. We also examined the effect of respiratory loading on diaphragm hypoxanthine concentrations to determine whether the stress of loading is sufficient to increase diaphragm levels of this xanthine oxidase pathway substrate.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Studies were conducted by using 36 male Sprague-Dawley rats. These animals were housed and cared for by the Case Western Reserve University Animal Resource Center according to Accreditation of Animal Laboratory Animal Care guidelines. Food and water were allowed ad libitum.
Decerebration procedure. Because loaded breathing is a harsh stress, we thought it unethical to examine the effects of this intervention in conscious animals. Moreover, the use of anesthesia distorts central motor outflow. For these reasons, animals were made decerebrate before the institution of respiratory loading in these experiments (22).
On the day of study, animals were anesthetized with ketamine (30 mg/kg im), supplemented with inhalational halothane delivered via face mask. A tracheostomy was performed, and ventilation was initiated by using a Harvard small-animal ventilator (Harvard Apparatus, South Natick, MA) set at tidal volume of 5 ml/kg and ventilation rate of 70 breaths/min. The right femoral artery was cannulated for the continuous monitoring of arterial blood pressure (by using a Gould pressure transducer; Gould Institute, Cleveland, OH) and to allow the removal of blood samples for arterial blood-gas measurements. Rats were then turned and placed in a prone position. The scalp was opened to expose the top of the skull. Two burr holes were made by using a small electric drill, and the craniotomy was enlarged by using bone rongeurs. By using a flat metal probe, an intercollicular decerebration was performed; the brain cephalid to this area was removed by using suction. Blood loss was controlled by gently packing this space with Gelfoam and cotton. After the decerebration, anesthesia was discontinued, and the animals were returned to a supine position. Mechanical ventilation was slowed to a rate of 15 breaths/min until spontaneous breathing efforts began (~3 breaths/min or fewer). At this time, rats were removed from the ventilator. Expiratory flow was monitored by using a pneumotachograph attached to the expiratory limb of a small Hans-Rudolph valve connected to the endotracheal tube. The signal from this tachograph was amplified and integrated (Charles Ward PI-830 integrator, CWE, Ardmore, PA) to yield tidal volume. Airway pressure was measured by means of a pressure transducer (model MP-45, Validyne, Northridge, CA) attached via a sidearm to the endotracheal tube. A small-gauge, water-filled polyethylene tube was inserted into the esophagus to monitor esophageal pressure. All of these signals were recorded continuously on a chart recorder (Gould Institute). Body temperature was monitored by using a rectal temperature probe and was maintained at 37°C by using a heat lamp.In vitro diaphragm force assessment. Diaphragm muscle used for in vitro force assessment was placed in a dish containing 95% O2-5% CO2-gassed Krebs-Henseleit solution (135 mM NaCl, 5 mM KCl, 11.1 mM dextrose, 2.5 mM CaCl2, 1 mM MgSO4, and 50 mg/l curare, adjusted to a pH of 7.40) (17). A small muscle strip was dissected and mounted vertically in a water-jacketed organ bath (Radnoti Glass, Monrovia, CA) containing Krebs-Henseleit solution and maintained at 27°C. The base of this strip was fixed to the bottom of the bath, and the central tendon portion of the strip was tied to a metal rod, which was suspended from a force transducer (model FT 10, Grass Instruments, Quincy, MA) mounted above the organ bath. A platinum field electrode was placed around the muscle strip to deliver electrical stimulation from a constant-current amplifier (Applied Neural Control Laboratories, Case Western Reserve University) attached to a Grass S48 stimulator. Strip length was then adjusted to the length at which twitch force was maximum. Amplifier current was adjusted so that it was ~20% greater than that required to obtain maximal force.
Exactly 20 min after the animal was killed, diaphragm contractile analysis was begun. Muscles were stimulated with 1-Hz impulses to assess twitch contraction and one-half relaxation times. After a 5-s rest, strips were then stimulated with trains of 1-, 10-, 20-, 50-, and 100-Hz impulses (train duration 800 ms) with a 5-s rest between adjacent trains. An additional 30-s rest was then provided, followed by a 5-min trial of repetitive, fatiguing, isometric contractions; i.e., 20-Hz trains of stimuli were applied at a train rate of 0.5 trains/s and with a train duration of 500 ms. At the completion of this repetitive contraction trial, strip length was measured, and muscles were removed from the bath and weighed.Hypoxanthine assay. Tissue hypoxanthine levels were measured in diaphragm samples by HPLC (9, 14). Muscle samples (100 mg) were first pulverized under liquid nitrogen and homogenized in 1 ml of ice-cold 3 M perchloric acid. Ice-cold ultrapure water was then added to the homogenate to bring the total volume to 5 ml. The suspension was centrifuged at 12,000 g for 20 min at 0°C. The resulting supernatant was removed and neutralized with 2 M KHCO3. After centrifugation at 3,000 g for 15 min at 0°C, 20 µl of the supernatant were injected onto a Waters µBondapak C18 HPLC column attached to a Varian 5000 gradient HPLC system (Varian Instruments, Sugar Land, TX). Samples were eluted from the column by using an acetonitrile gradient of 1-50% in KH2PO4 at a flow rate of 0.7 ml/min. Absorbance at 254 nm was monitored continuously. Sample peaks were analyzed by using a Hewlett-Packard 3390A integrator. Hypoxanthine concentrations were calculated by comparing sample peak areas to the peaks obtained from known amounts of hypoxanthine standards run under identical conditions.
Oxypurinol assay. Plasma oxypurinol levels were determined by HPLC methods according to Dorion et al. (8). Plasma (0.5 ml) was added to 0.2 ml of 20% perchloric acid, mixed well, and centrifuged at 8,500 g for 10 min at 4°C. The supernatant was removed and filtered through a 22-µm Millipore GV13 syringe filter. A 20-µl aliquot of the filtrate was analyzed by using a Varian 5000 HPLC machine, operating in isocratic mode, equipped with a Waters µBondapak C18 column, an absorbance detector set at 254 nm, and a Hewlett-Packard 3390A integrator. The mobile phase was 50 mM KH2PO4, pH 6.0, at a flow rate of 1 ml/min. Authentic oxypurinol standards were run under similar conditions, and these values were used to calculate concentrations of oxypurinol in plasma samples.
Determination of lipid peroxidation. Lipid peroxidation was assessed by measuring diaphragm thiobarbituric acid-reactive substances (TBARS) levels. This is a commonly used but nonspecific measure of lipid peroxidation. In general, this index tends to rise and fall in concert with more specific indexes of free radical-induced lipid and protein modification. In the diaphragm, we have recently demonstrated that tissue levels of TBARS rise during fatiguing contraction. Moreover, free radical scavenger administration appears to ablate contraction-related increases in TBARS in parallel with an effect of these agents to reduce fatigue rate (26).
The TBARS assay was performed in keeping with previous reports (11, 13). In brief, 100 mg of muscle were homogenized in cold 1.15% KCl to yield a 10% solution (wt/vol). A portion of this homogenate (0.2 ml) was added to a tube containing 0.2 ml of 8.1% sodium dodecyl sulfate, 1.5 ml of 20% acetic acid (pH 3.5), and 1.5 ml of 0.8% thiobarbituric acid. The mixture was made up to 4 ml with distilled water, capped, and heated at 95°C for 60 min. After it cooled to room temperature, 5 ml of n-butanol and 1 ml of distilled water were added. The mixture was centrifuged at 2,500 rpm for 20 min, at which time the supernatant was removed and read in a spectrophotometer (Shimadzu, Kyoto, Japan) at 532 nm. Absorbance values were converted to TBARS concentrations by comparison to a standard curve constructed by using known amounts of tetramethoxypropane. Diaphragm TBARS values were expressed as nanomoles per gram of tissue.Experimental protocol. In previous studies Supinski et al. (21, 25) have observed differences in the load response of decerebrate animals breathing supplemental oxygen as opposed to those breathing room air. Most importantly, they found that administration of N-acetylcysteine, a free radical scavenger, improves respiratory performance during loaded breathing performed with animals receiving supplemental oxygen but did not alter performance for animals loaded but breathing room air (24, 25). These previous data suggest that the importance of oxygen-derived free radicals in modulating respiratory muscle dysfunction and respiratory failure during loading may vary as a function of loading conditions and the concentration of inspired oxygen. For this reason, we chose to study both room air breathing and 100% oxygen-supplemented decerebrate animals. In all, eight groups of rats were studied: 1) unloaded, saline-treated animals breathing room air; 2) unloaded oxypurinol-treated animals breathing room air; 3) loaded, saline-treated animals breathing room air; 4) loaded, oxypurinol-treated animals breathing room air; 5) unloaded, saline-treated animals breathing 100% oxygen; 6) unloaded, oxypurinol-treated animals breathing 100% oxygen; 7) loaded, saline-treated animals breathing 100% oxygen; and 8) loaded, oxypurinol-treated animals breathing 100% oxygen. When called for, oxypurinol (50 mg/kg bid) was administered on the day before study; an additional 50 mg/kg were given 30 min into the equilibration period. This procedure follows that of Dorion et al. (8) and Zimmerman et al. (29) and results in plasma oxypurinol concentrations that have been reported to effectively inhibit xanthine oxidase activity without producing a scavenging effect.
On the day of study, decerebration and instrumentation were carried out as described in Decerebration procedure. Once this was completed, a 1-h equilibration period was provided to allow any remaining anesthesia to wear off. At the conclusion of this equilibration period, an arterial blood sample was drawn, and loading was initiated. Inspiratory resistive loads of 5,000 and 17,000 cmH2O · l
1 · s
were applied to the loaded, room-air-breathing groups and the loaded,
100% oxygen-breathing groups, respectively. These levels
of load were chosen on the basis of previous work in our laboratory
indicating that resistances of these magnitudes result in task failure
in ~60 min in nonoxypurinol-treated animals (i.e., a higher load is
required in oxygen-treated animals to reach this end point in 60 min)
(unpublished observations). Peak inspiratory airway
pressure was achieved after ~3 min in loaded breathing trials, and
this level of pressure was recorded; loading was then continued until
the airway pressure had fallen by 50% from this peak value. At that
time, a second arterial blood-gas sample was drawn, and a blood sample
for plasma oxypurinol level was taken. Animals were then killed by the
administration of pentobarbital sodium. The entire
diaphragm was quickly removed (within 60 s in all cases); one portion
of the diaphragm was used for in vitro force assessment, and the
remaining muscle was frozen in liquid nitrogen and stored for
biochemical analyses. Nonloaded control groups underwent a similar
protocol, save that no load was applied and physiological monitoring
was continued for 1 h after completion of the equilibration period.
Statistical analysis. A one-way ANOVA was used to compare single variables (e.g., hypoxanthine levels) across animal groups, with post hoc testing (Student-Newman-Keuls) used to determine statistical differences between individual groups.
A repeated-measures ANOVA was used for comparisons in which repeated measurements of a given variable were made under different conditions (e.g., force-frequency curves from different groups). Data are presented as means ± SE. A P value < 0.05 was taken to indicate statistical significance.| |
RESULTS |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Loaded breathing trials.
Inspiratory pressure generated during loaded breathing is displayed in
Fig. 1 for both room-air-breathing and
100% oxygen-breathing groups. After an initial immediate rise after
the application of loads, inspiratory pressures progressively fell
during trials in all groups of loaded animals. Oxypurinol
administration had no effect on the rate of these declines in either
room-air-breathing or oxygen-supplemented rats. Figure
2 shows inspiratory tidal volume over time
for loaded breathing studies; oxypurinol administration also had no
effect on this parameter. Similarly, oxypurinol administration had no
effect on breathing pattern (Table 1) or
arterial blood gases (Table 2).
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In vitro diaphragm force generation.
Diaphragm in vitro force-frequency curves are illustrated in Fig.
4. Loading resulted in decreases in
diaphragm force generation at all stimulation frequencies compared with
muscles removed from unloaded animals in both room-air-breathing groups
(P < 0.01) and in 100%
O2-breathing groups
(P < 0.01). Oxypurinol
administration failed to improve force production. In fact,
force-frequency curves from oxypurinol-treated loaded animals were
slightly lower than those from their non-oxypurinol-treated
counterparts.
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Hypoxanthine levels.
Tissue hypoxanthine concentrations for all groups are displayed in Fig.
5. Oxypurinol administration to loaded
animals breathing supplemental oxygen was not associated with increases
in muscle hypoxanthine levels compared with levels for loaded animals
injected with saline. In room-air-breathing animals, however,
oxypurinol did have an effect on muscle hypoxanthine concentrations,
which were nearly twice as high in the oxypurinol, loaded group
compared with the saline-treated loaded group
(P < 0.03).
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Oxypurinol levels.
Oxypurinol levels were measured on plasma samples obtained at the end
of studies to ensure that effective drug concentrations were present.
These data are presented in Fig. 6.
According to previously published literature (8), blood levels of 1.3 µg/ml (equivalent to 10 µM) are required to achieve >80%
inhibition of xanthine oxidase activity. Each of the groups in this
study to which oxypurinol was administered yielded concentrations in excess of this value. Oxypurinol levels in loaded animals were slightly
lower than in unloaded controls but were still greater than twice the
concentration required for enzyme inhibition.
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TBARS levels.
Diaphragm TBARS levels, a marker of lipid peroxidation, were
significantly higher in loaded animals than in unloaded animals for
room-air-breathing trials (Fig.
7A;
P < 0.01 saline loaded vs. saline
unloaded and P < 0.01 for oxypurinol
loaded vs. oxypurinol unloaded). Diaphragm TBARS were also higher for
loaded animals receiving supplemental oxygen (Fig.
7B; P < 0.01 for saline loaded vs. saline unloaded and
P < 0.001 for oxypurinol loaded vs.
oxypurinol unloaded). Within the loaded groups, oxypurinol did not
significantly reduce the amount of TBARS formation.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Comparison to previous reports. Several recent reports have indicated that strenuous diaphragmatic contraction is associated with a significant increase in free radical formation within this muscle. As evidence of this assertion, several reports have found direct evidence of production of several radical species by or in the contracting diaphragm (i.e., formation of superoxide anions, hydroxyl radicals, generation of hydrogen peroxide) (4, 15, 16). Other work has found indirect evidence of free radical generation during muscle contraction, reporting increases in tissue levels of protein oxidation and lipid peroxidation by-products (i.e., TBARS formation, increases in isoprostane concentrations, reductions in GSH, and increases in GSSG, formation of protein carbonyls) (2, 23, 26). These phenomena have been reported with in vitro electrically stimulated muscle strips, in situ electrically contracting muscle, and in vivo after elevations of the diaphragmatic workload induced by applying large resistive loads to inspiration (2, 15, 16, 23, 24, 26). Importantly, these studies have also suggested that the free radicals produced during strenuous muscle contractions have significant functional consequences. In support of this concept, several groups have shown that administration of free radical scavengers to electrically stimulated muscle reduces the rate of development of muscle fatigue (12, 15, 26).
The data we collected for our "control" animal groups in the present study (i.e., loaded and unloaded animal groups not treated with oxypurinol) are consistent with these past reports. In the control groups that underwent resistive loaded breathing, we found evidence of both lipid peroxidation within the diaphragm and of a significant reduction in diaphragmatic force generation at the end of loaded breathing trials compared with measurements made in unloaded control animals. These latter data indicate, moreover, that the regimen of loading employed in the present study was such that significant diaphragmatic fatigue was induced.Potential pathways of free radical formation during loaded breathing. Although the observations made in the above-referenced studies constitute compelling evidence that free radicals play an important role in modulating muscle function during strenuous contractions, the precise mechanism by which contraction elicits increases in radical formation has not been established. Certainly, a number of cellular pathways are capable of generating superoxide anions in muscle cells, including the cytochrome P-450 system, the cyclooxygenase-mediated conversion of PGH2 to PGI2, the mitochondrial electron transport chain, and the xanthine oxidase catalyzed metabolism of hypoxanthine (20).
A number of previous reports have argued that the last of these pathways (i.e., superoxide production as a by-product of xanthine compound metabolism) is probably the most important source of free radicals in contracting skeletal muscle (1, 3, 5). Specifically, Barclay and Hansel (3) reported that administration of allopurinol, a xanthine oxidase inhibitor, reduced the rate of development of muscle fatigue in the dog hindlimb, acting in a fashion similar to the administration of a free radical scavenger (i.e., DMSO). They interpreted this effect as indicating a potential role for xanthine oxidase-mediated superoxide formation in the generation of free radicals in the muscle being studied (3). In addition, Andrade et al. (1) found that animals fed a diet deficient in selenium, a metal required for xanthine oxidase activity, developed less diaphragm fatigue when subjected to inspiratory resistive loading and manifested less diaphragm glutathione oxidation than did control animals fed a normal diet (1). These data are consistent with a potential role for xanthine oxidase in modulating free radical formation in the respiratory muscles during loaded breathing and would seem to suggest that free radical production was reduced in the selenium-depleted animals. The present study was designed to provide a direct test of the effect of inhibiting xanthine oxidase (by the administration of oxypurinol) on diaphragm function during loaded breathing. If xanthine oxidase-mediated formation of free radicals plays an important role in modulating the rate of development of diaphragm fatigue during respiratory loading, then oxypurinol administration should have either 1) increased the ability of animals to tolerate the applied load (i.e., pressure and volume generation would be enhanced and a longer time would be required to develop evidence of task failure), 2) reduced the amount of diaphragm dysfunction present at the end of the loaded breathing trial, 3) reduced the amount of lipid peroxidation associated with loaded breathing, or 4) produced some combination of these findings. Instead, we found that the regimen of oxypurinol administered in this study failed to influence load tolerance or alter the degree of diaphragm dysfunction resulting from inspiratory loaded breathing. Although there was a trend for our index of lipid peroxidation (i.e., TBARS) to be partially suppressed by oxypurinol administration in room-air-breathing loaded animals, this effect did not reach statistical significance. This trend, however, was only for partial suppression of loading-induced increases in TBARS, and this index remained higher in the oxypurinol-treated group of loaded animals than for unloaded saline-treated control studies. One possible explanation for our findings is that free radicals are formed by this pathway under these conditions, but the degree to which free radicals influence muscle function in these circumstances is so small that an effect to suppress radical formation had no functional consequences. We think this possibility is unlikely, because the animal model and loading conditions tested in the present experiment were identical to those used in a previous experiment performed by Supinski et al. (24), who found that administration of N-acetylcysteine, a free radical scavenger and glutathione precursor, slowed the rate of development of respiratory failure (i.e., better preserved pressure generation by the respiratory muscles over time during a sustained period of respiratory loading) and prolonged the time to respiratory arrest in inspiratory resistive -loaded animals. These previous results suggest that radical- induced muscle dysfunction is functionally important and may modulate the course of development of respiratory failure during loaded breathing under the conditions tested in the present study. If so, it would argue that the failure of oxypurinol to improve respiratory system performance in the present study was likely a consequence of a failure of this agent to adequately inhibit free radical formation. The fact that oxypurinol administration did not prevent the development of lipid peroxidation, i.e., did not ablate loading-related increases in TBARS in the present study, is also of interest. In previous work, we have shown that doses of free radical scavengers that reduce the rate of development of diaphragmatic muscle fatigue also completely suppress formation of TBARS in the diaphragm (26). The failure of oxypurinol administration to prevent increases in TBARS concentrations is further evidence, therefore, that this agent failed to adequately suppress free radical formation in the diaphragm during respiratory loading.Oxypurinol dosage. We should also consider the possibility that the dosage of oxypurinol administered in this study may have been too small and that significant effects of this agent might have been observed if we had used higher doses. The dosage employed has, however, been used in previous studies that have demonstrated easily detectable effects on other forms of tissue injury that are known to be mediated by free radicals formed by the xanthine oxidase pathway (8, 19). Moreover, we measured blood oxypurinol levels and found these to be in a range that should have been sufficient to suppress xanthine oxidase-mediated free radical formation (8).
We would also argue that the fact that hypoxanthine levels were different in room-air-breathing oxypurinol-treated and saline-treated loaded groups is evidence of xanthine oxidase inhibition by oxypurinol in our loaded groups of animals. One would not expect breathing to be much of a metabolic stress to the respiratory muscles in the absence of loading, so it is no surprise that hypoxanthine levels were not be increased in nonloaded groups in either the absence or presence of oxypurinol. Specifically, at the low levels of metabolic activity associated with nonloaded breathing it is probable that normal metabolic pathways (i.e., oxidative phosphorylation) were sufficient to regenerate ATP from ADP and that breakdown of ADP to adenosine via the xanthine dehydrogenase/oxidase pathways was not significant. During loading, far greater ATP usage would be expected, increasing the probability of hypoxanthine formation; loading in the presence of oxygen limitation (i.e., inhalation of room air without supplemental oxygen) would be even more likely to stress the metabolic machinery, increasing hypoxanthine formation. Our findings are consistent with this possibility, with hypoxanthine levels elevated specifically in the oxypurinol-treated, room-air-breathing, loaded group of animals (see Fig. 5). The fact that hypoxanthine levels were not elevated in the group of room-air-breathing animals undergoing loading in the absence of oxypurinol is consistent with our contention that the dose of oxypurinol used in this study was able to inhibit xanthine oxidase. If this dose had not inhibited xanthine oxidase, the hypoxanthine levels in the saline- and oxypurinol-treated room-air-breathing loaded groups should have been exactly the same. As best we can see, there is no way to explain these latter results other than to conclude that oxypurinol specifically inhibited hypoxanthine breakdown by xanthine oxidase in our loaded, room-air-breathing groups of animals. Because the same dosage of oxypurinol was used for oxygen-treated loaded groups, the most likely explanation for the failure of hypoxanthine to rise in the oxypurinol-treated, oxygen-breathing, loaded group is that the metabolic stress provided by this latter regimen of testing was insufficient to appreciably increase flux of ADP down the degradation pathway.Implications. One might ask whether it is reasonable to postulate, a priori, that one pathway of oxygen free radical generation is dominant in a complex stress such as respiratory loading. Such a possibility is supported by the fact that studies of other conditions (i.e., myocardial infarction, cerebral ischemia) demonstrate that inhibition of specific pathways of free radical formation can have large functional consequences. For example, administration of xanthine oxidase inhibitors to rat heart results in pronounced protection from the effects of ischemia-reperfusion. Although the xanthine oxidase pathway may be very important in generating free radicals in the ischemic rat heart (27), the present data indicate that this pathway does not appear to mediate free radical formation under the conditions of respiratory loading examined in the present study.
If free radical formation in the diaphragm during loaded breathing is not the result of a xanthine oxidase- catalyzed reaction, one must ask why Andrade et al. (1) found that rats depleted of selenium, an essential cofactor for xanthine oxidase activity, developed less diaphragmatic fatigue during inspiratory resistive loading. One possibility is that selenium depletion has effects on diaphragm function that confer some form of fatigue resistance that is entirely unrelated to inhibition of xanthine oxidase activity. This metal is a cofactor for a number of other enzyme systems, including aldehyde dehydrogenase, and it is conceivable that selenium depletion significantly alters muscle functional properties by affecting these other metabolic pathways. Another possibility is that the loading conditions examined by Andrade et al. (1) were sufficiently different from those examined in the present experiment that free radicals were generated by different mechanisms in the two experiments. As mentioned earlier, there are a number of potential mechanisms of free radical generation in muscle, including generation of superoxide by cyclooxygenase pathways, by the mitochondrial electron transport chain, as a by-product of phospholipase A2 metabolism, by nitric oxide synthase, and by the cytochrome P-450 system (20). It seems possible that several pathways of free radical formation participate in radical formation in muscle, with the importance of a specific pathway varying from condition to condition. For example, whereas diaphragm hypoxanthine levels were relatively low in the present study, previous work has suggested that this substrate for the xanthine oxidase pathway can increase to very high levels in other physiological situations (e.g., during hemorrhagic shock) (10). It seems possible that some conditions (including some forms of respiratory loading) that concomitantly stress the cardiovascular and respiratory systems may elicit xanthine oxidase-mediated diaphragmatic free radical generation, whereas other conditions that primarily act to load the respiratory muscles (i.e., the situation examined in the present experiment) do not. In summary, this study demonstrated that the inhibition of xanthine oxidase by oxypurinol did not improve diaphragm performance during inspiratory resistive- loaded breathing. Compared with controls, load tolerance and in vitro force generation were unaltered in oxypurinol-treated animals despite the fact that high concentrations of oxypurinol were present in the plasma of oxypurinol-treated loaded rats. Oxypurinol administration also failed to completely suppress formation of TBARS in the diaphragms of loaded animals. In light of these results, further studies need to be conducted to determine the primary source or sources of oxygen-derived free radical generation in the diaphragm during resistive loaded breathing.| |
ACKNOWLEDGEMENTS |
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This work was supported by National Heart, Lung, and Blood Institute Grants HL-54825 and HL-38926.
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FOOTNOTES |
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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.
Address for reprint requests and other correspondence: G. Supinski, MetroHealth Medical Center, 2500 MetroHealth Dr., Cleveland, OH 44109.
Received 1 July 1998; accepted in final form 10 May 1999.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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