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Am. J. Respir. Crit. Care Med., Volume 165, Number 3, February 2002, 412-418

Molecular Characterization of a Superoxide-Generating NAD(P)H Oxidase in the Ventilatory Muscles

DANESH JAVESGHANI, SHELDON A. MAGDER, ESTHER BARREIRO, MARK T. QUINN, and SABAH N. A. HUSSAIN

Critical Care and Respiratory Divisions, Department of Medicine, Royal Victoria Hospital and Meakins-Christie Laboratories, McGill University, Montreal, Quebec, Canada; and Veterinary Molecular Biology, Montana State University, Bozeman, Montana


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The molecular sources of reactive oxygen species (ROS) in skeletal muscles are not well understood. We hypothesized that nonphagocyte NAD(P)H oxidase could be a source of ROS in muscle fibers. We thus investigated the existence, structure, and contribution of nonphagocyte NAD(P)H oxidase to ROS production in rat skeletal muscles. ROS production and NAD(P)H oxidase activity were evaluated by lucigenin-enhanced chemiluminescence and NADH consumption rate, whereas enzyme composition was monitored by reverse transcription-polymerase chain reaction and immunoblotting. Basal O2- production in muscle strips from normal rats averaged 1.4 nmol/mg per 10 min and increased to ~ 18 nmol/mg per 10 min in the presence of NADH. Muscle O2- production and NADH consumption were inhibited by Tiron, superoxide dismutase, apocynin, and diphenyleneiodonium but not by inhibitors of cyclo-oxygenases, xanthine oxidase, nitric oxide synthases (NOS), and mitochondrial enzymes. We detected mRNA and proteins of p22phox, gp91phox, p47phox, and p67phox subunits in normal rat muscles. These subunits were localized in close proximity to the sarcolemma. Induction of sepsis in rats doubled muscle O2- production with no major changes in muscle NADPH oxide subunit expression. In lipopolysaccharide-treated but not in control muscles, O2- production was increased significantly by NOS inhibition. We conclude that a constitutively active NAD(P)H oxidase enzyme complex exists in normal skeletal muscle fibers and contributes to ROS production. In septic rats, this production is increased but measurable O2- is reduced by enhanced NO production.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Keywords: NADPH oxidase; nitric oxide; oxygen radicals; sepsis; skeletal muscle

Reactive oxygen species (ROS) in general and superoxide anions (O2-) in particular are produced inside skeletal muscle fibers under normal and pathological conditions (1, 2). In resting skeletal muscles, O2- radicals are released into the extracellular space at relatively low rates, and the rate of O2- production rises significantly in response to increased muscle activity (3, 4). Many cellular sources are involved in the production of ROS, including NAD(P)H-dependent electron transport chains, membrane-bound oxidoreductases, cytosolic xanthine oxidase, and the cyclo-oxygenase pathway of arachidonic acid metabolism. The contribution of these sources to O2- production has been investigated mainly in lung tissue, hepatic tissues, endothelial cells, and vascular smooth muscles, but the exact involvement of these sources in ROS production inside skeletal muscle fibers remains unclear. Studies suggest that a significant portion of ROS production in response to increased muscle activity is not derived from the mitochondria and is dependent on the presence of NAD(P)H (5). These results suggest that a nonmitochondrial oxidase, which uses NADPH, exists in skeletal muscles and increases its production of ROS in response to increased muscle activity.

NAD(P)H oxidase, which catalyzes the production of O2- by one-electron reduction of O2, using NADPH or NADH as the electron donor, exists both in phagocytes (6) and in nonphagocytes such as fibroblasts, chondrocytes, and mesangial, microglial, epithelial, endothelial, and vascular smooth muscle cells (for review, see Reference 7). Significant differences have emerged between phagocytic and nonphagocytic NAD(P)H oxidases such as those concerning subunit structure, substrate preference, and time course of activity (7). Despite progress in the characterization of NAD(P)H oxidase in vascular and mesangial cells, no information is yet available regarding the existence, subunit structure, localization, and contribution of this enzyme system to the production of O2- in skeletal muscle fibers.

Nitric oxide (NO) is normally synthesized inside skeletal muscle fibers by the neuronal (nNOS) and endothelial (ecNOS) nitric oxide synthases (8, 9). Accumulating evidence indicates that NO modulates intracellular ROS levels in vascular cells through a direct interaction with O2- and indirectly through its effect on enzymatic activities responsible for ROS production, such as that of NAD(P)H oxidase and xanthine oxidase (10, 11). Despite the presence of abundant NOS proteins inside skeletal muscle fibers, little is known about the influence of NO on O2- production in these fibers.

We hypothesized in this study that NAD(P)H oxidase exists inside skeletal muscle fibers and contributes to the production of ROS inside these fibers. To test this hypothesis, we studied subunit composition, localization, and substrate use of the NAD(P)H oxidase enzyme complex system in normal skeletal muscles. We also measured the contribution of NAD(P)H oxidase to skeletal muscle ROS production under normal conditions and in response to severe sepsis. Finally, we investigated whether constitutive NO release inside muscle fibers influences the production of O2- by muscle NAD(P)H oxidase.

    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

More details are provided in the online data supplement.

General Animal Preparation

Adult male Sprague-Dawley rats were killed with an overdose of pentobarbital sodium and the diaphragm and limb muscles were either excised and frozen in liquid nitrogen or embedded in Histo Prep medium, frozen in cooled isopentane, and then stored at -80° C. In a few experiments, muscles were removed and quickly placed in ice-cold Krebs-HEPES buffer, and then radial strips were excised from each muscle and placed in the same buffer for a period of 30 min.

Induction of Sepsis

Two groups (n = 6 in each group) of male rats were examined 18 h after intraperitoneal injection of either normal saline (control group) or Escherichia coli lipopolysaccharide (LPS, serotype 055:B5; 10-12 mg/kg) (septic group). We also examined five groups (n = 6 in each group) of rats, which were killed 1, 3, 6, 12, and 24 h after LPS injection and from which the diaphragms were collected as mentioned above.

Preparation of Muscle Fractions

Separation of mitochondrial, membrane, and cytosolic muscle fractions was performed as described by Rock and coworkers (12). In brief, crude muscle homogenates were prepared by homogenization in a specific buffer (12) and were then centrifuged at 1,000 × g. The mitochondrial fraction was collected from crude homogenates by centrifugation at 12,000 × g. The resulting supernatants then underwent centrifugation at 37,500 × g, yielding supernatants (cytosolic fraction) and pellets (membrane fraction). The Bradford method (Bio-Rad, Hercules, CA) was used to measure protein levels in all muscle fractions.

Measurement of O2- Radicals by Lucigenin-Derived Chemiluminescence

Increasing xanthine levels were incubated with cytochrome c (80 µM) and 8 units of xanthine oxidase at 37° C in the presence and absence of superoxide dismutase (SOD) (1.5 U/ml). Absorbance at 550 nm was measured with a spectrophotometer and the levels of O2- produced were calculated as described previously (13). The amount of O2- calculated was then used to calibrate lucigenin-derived cheniluminescence (LDCL) signal obtained by mixing the same levels of xanthine and xanthine oxidase in a luminometer (Lumat LB 9501; Berthold, Pforzheim, Germany). For measurement of O2- in muscle samples, muscles were heated in a water bath to 37° C and lucigenin (230 µM) was then added to the tube, which was then immediately placed inside the luminometer. Luminometer output was measured for a 10-min period. Muscle LDCL was measured in the presence of lucigenin alone (basal), NADH (100 µM), and NADPH (100 µM). Additional groups of diaphragmatic muscle strips were preincubated for 20 min at 37° C with Tiron (10 mM), diphenyleneiodonium (DPI, 1.85 µM), SOD (0.5 U/ml), NG-nitro- L-arginine methyl ester (L-NAME, 1 mM), oxypurinol (300 µM), indomethacin (100 µM), and rotenone (250 µM). Basal and NADH-stimulated LDCL signals were then measured as mentioned above.

Measurement of NADH Consumption

Aliquots of muscle fractions (12.5 to 100 µg of total proteins) were incubated with NADH (100 µM) at 37° C in the absence and presence of the above-mentioned antioxidants and inhibitors of ROS-generating enzymes and the rate of NADH consumption was monitored by measuring the decline in absorbance at lambda  = 340 nm.

Reverse Transcription-Polymerase Chain Reaction

Muscle total RNA (1 µg) was reverse transcribed with random hexamers and Moloney murine leukemia virus (Mo-MuLV) reverse transcriptase (RT). Rat-specific NADH oxidase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNAs were then amplified by polymerase chain reaction (PCR) using specific oligonucleotide primers (Table 1). Ethidium bromide-stained 2% agarose gels and an optical density scanner were used to separate, visualize, and quantify the intensity of PCR products.

                              
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TABLE 1

 MOLECULAR SEQUENCE OF RT-PCR OLIGONUCLEOTIDE PRIMERS USED TO AMPLIFY mRNA OF VARIOUS NADPH OXIDASE SUBUNITS IN RAT SKELETAL MUSCLES*

Immunoblotting

Muscle fractions (80 µg per sample) were mixed with sample buffer, boiled, loaded onto Tris-glycine sodium dodecyl sulfate (SDS) polyacrylamide gels, and separated by electrophoresis. Lysates of human and rat neutrophils were used as positive controls. Proteins were transferred electrophoretically to polyvinylidene difluoride (PVDF) membranes, blocked with nonfat dry milk, and then incubated with two sets of primary monoclonal or polyclonal anti-human NAD(P)H oxidase antibodies (14). Membranes were then washed and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies. Specific proteins were detected with a chemiluminescence kit.

Immunohistochemistry

Frozen tissue sections were fixed with acetone, rehydrated with phosphate-buffered saline (PBS), blocked with normal donkey serum, and then incubated overnight with anti-human NAD(P)H oxidase antibodies. For negative control, primary antibodies were replaced with nonspecific mouse or rabbit IgGs. After three rinses with PBS, sections were incubated with Cy3-labeled anti-mouse or anti-rabbit secondary antibodies, washed, mounted with coverslips, and examined with a fluorescence microscope.

Statistical Analysis

Values are presented as means ± SEM. Differences in O2- production, protein optical densities, and rate of NADH use were detected by one-way analysis of variance (ANOVA) followed by the Tukey test for multiple comparisons. Linear regression analysis was used to quantify the rate of NADH consumption in the absence and presence of various inhibitors.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Production of O2- Radicals in Normal Skeletal Muscles

Luminometer output was linearly related to the amount of xanthine used during the production of the calibration curve (ranging between 1 and 64 nmol) (r = 0.99; Figure 1A). The addition of SOD resulted in a signal that was not different from background, confirming that O2- radicals are responsible for the luminescence. Figure 1B shows representative LDCL signals obtained from normal rat diaphragms. LDCL signals were significantly higher in the presence of NADH than under basal conditions or in the presence of NADPH (Figure 1B). Figure 2 shows mean values of O2- radicals produced by normal rat diaphragms. Muscle O2- levels were significantly higher in the presence of NADH compared with basal levels and those measured in the presence of NADPH (Figure 2A). Basal O2- levels were significantly attenuated by SOD, DPI, Tiron, and oxypurinol (p < 0.05), whereas L-NAME, indomethacin, and rotenone were without any significant effects on basal O2- levels (Figure 2B). Similarly, NADH-enhanced O2- radical production was significantly reduced by SOD, DPI, and Tiron but not by L-NAME, oxypurinol (Figure 2C), and rotenone (not shown). The observation that DPI inhibited both basal and NADH-dependent O2- production, whereas only the basal O2- level was attenuated by oxypurinol, suggests that both xanthine oxidase and NAD(P)H oxidase contribute to basal ROS production, whereas NAD(P)H oxidase is the main source of NADH-stimulated ROS production in normal muscle fibers. A similar preference of muscle O2- production for NADH was also found in the soleus and gastrocnemius muscles (Figure 3).


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  		Figure 1.   (A) In vitro LDCL signals generated in response to increasing levels of xanthine (1 to 32 nmol) in the presence of 8 units of xanthine oxidase. Note that increasing xanthine levels elicited progressively greater rises in integrated LDCL signals. Insert: Relationship between the area under the chemiluminescence curve (integral) and the level of xanthine. (B) Representative examples of LDCL signals generated by normal rat diaphragmatic strips under basal conditions and in the presence of NADH or NADPH. Note that NADH elicited a significantly greater luminescence than that generated in the presence of NADPH.


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  		Figure 2.   (A) Production of O2- radicals by normal rat diaphragmatic strips in the absence (basal) or presence of either NADH (100 µM) or NADPH (100 µM). *p < 0.05 and **p < 0.01 compared with basal condition. (B) The influence of various inhibitors of ROS-producing enzymes on basal O2- production in normal rat diaphragmatic strips. *p < 0.05 compared with control (no inhibitors). Note that SOD, DPI, Tiron, and oxypurinol significantly attenuated basal O2- production. (C ) Effects of inhibitors of ROS-producing enzymes on NADH-enhanced O2- production in normal rat diaphragmatic muscle strips. *p < 0.05 compared with control (no inhibitors).


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  		Figure 3.   Production of O2- radicals under basal conditions and in the presence of either NADH (100 µM) or NADPH (100 µM) in intact diaphragmatic, gastrocnemius, and soleus muscle samples of normal rats. Note that in the three muscles, NADH-enhanced O2- production was much greater than that elicited by NADPH.

NADH Consumption

No significant NADH consumption was detected in cytosolic muscle fractions even at protein aliquots exceeding 100 µg. By comparison, NADH consumption by the membrane fraction rose linearly with respect to time at protein levels of 12.5 and 25 µg (Figure 4A). However, at 50 and 100 µg, NADH consumption peaked within 40 and 10 min, respectively (Figure 4A). As shown in Figure 4B, the rate of NADH consumption by the membrane fraction of normal rat diaphragm rose linearly with sample protein concentration (r = 0.95, p < 0.001). The NADH consumption rate (measured at the 50-µg total protein level over a 40-min period) of the diaphragmatic membrane fraction was significantly inhibited by apocynin and DPI (p < 0.05 and p < 0.01, respectively) but not by indomethacin, oxypurinol, rotenone, or L-NAME (Figure 5).


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  		Figure 4.   (A) In vitro NADH consumption by isolated normal rat diaphragmatic membrane fractions versus time with different amounts of sample proteins. Note that whereas NADH consumption continues to increase at sample protein levels of 12.5 and 25 µg, NADH consumption peaked within 40 and 10 min when sample protein levels were 50 and 100 µg, respectively. (B) The relationship between NADH consumption rate of normal diaphragmatic muscle membrane fraction and total sample protein levels (r = 0.95, p < 0.001).


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  		Figure 5.   Effects of inhibitors of various ROS-producing enzymes on in vitro NADH consumption rate of normal rat diaphragmatic membrane fractions. *p < 0.05 and **p < 0.01 compared with control (no inhibitors). Note that only DPI and apocynin significantly attenuated NADH consumption, whereas other inhibitors were without effect.

NAD(P)H Oxidase Subunit Structure and Localization

We detected mRNA transcripts of p22phox, gp91phox, p47phox, and p67phox subunits of phagocyte NADPH oxidase in diaphragmatic and gastrocnemius samples, using RT-PCR (Figure 6). The sequences of partial cDNAs of muscle p22phox and gp91phox were identical to those found in other rat organs (17, 18). We also found that partial cDNAs of muscle p47phox and p67phox subunits have relatively higher homology with mouse neutrophil sequences than with human sequences (Appendices 1 and 2; see online data supplement). In addition to mRNA transcripts, we were able to detect p22phox, gp91phox, p47phox (Figure 7A), and p67phox proteins in normal rat muscles. Interestingly, muscle p22phox protein had an apparent mass of about 30 kDa, which is larger than reported for neutrophil NADPH oxidase subunit. This difference is not related to species differences between humans and rats because the anti-p22phox antibodies detected a 22-kDa protein in rat neutrophil lysate (not shown). Muscle p22phox protein is localized in the membranous rather than in the mitochondrial and cytosolic fractions (Figure 7B). Similarly, we detected gp91phox and p47phox proteins in the membranous fraction of muscle homogenates (results are not shown). We were unable to detect p40phox protein in muscle samples despite the use of two separate monoclonal antibodies. Abundant positive p22phox immunostaining was detected inside skeletal muscle fibers in close proximity to the sarcolemma (Figure 8, top). Nerve fibers did not positively stain with anti-p22phox antibodies. Similar patterns of muscle fiber- and blood vessel-specific immunoreactivity were detected with anti-gp91phox, p47phox, and p67phox antibodies (Figure 8). Positive p40phox immunoreactivity was detectable only in blood vessels traversing muscle fibers. Negative control samples showed no prominent positive immunoreactivity.


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  		Figure 6.   RT-PCR products of mRNA of various NAD(P)H oxidase subunits in normal skeletal muscles.


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  		Figure 7.   (A) Immunoblotting of crude homogenates for rat muscles with antibodies selective for NAD(P)H oxidase subunits. Human neutrophil lysates were used as positive controls. Note that the relative mass of p22phox protein in rat muscle samples was larger than that in human neutrophils. (B) Immunoblotting of membrane, mitochondrial, and cytosolic fractions of normal rat diaphragm with anti-p22phox antibody. Note that p22phox protein was abundantly expressed in the membrane fraction rather than in the mitochondrial or cytosolic fractions.


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  		Figure 8.   Localization of p22phox (gastrocnemius), gp91phox (soleus), p67phox (gastrocnemius), p47phox (soleus), and p40phox (soleus) NAD(P)H oxidase subunits in normal rat skeletal muscle samples. Note that whereas p22phox, gp91phox, p67phox, and 47phox were localized inside muscle fibers in close proximity to the sarcolemma, p40phox subunit was present exclusively in blood vessels.

Effects of Sepsis on Muscle NAD(P)H Oxidase and O2- Production

No significant alterations in protein expression of p22phox, gp91phox, and p47phox subunits were observed up to 24 h after LPS injection, whereas p67phox subunit expression rose significantly higher than control values only after 24 h of LPS injection (Figure 9A). Basal diaphragmatic O2- levels (measured after 18 h of experimental period) were significantly higher in the LPS group compared with control muscles (p < 0.05; Figure 9B), whereas NADH-induced O2- production was similar in the two groups (Figure 9B). However, whereas L-NAME had no effects on NADH-stimulated O2- production in control diaphragm, L-NAME significantly elevated NADH-induced O2- production in septic diaphragms (p < 0.05 compared with NADH alone) (Figure 9B).


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  		Figure 9.   (A) Influence of sepsis on protein expression of various subunits of NAD(P)H oxidase of rat diaphragm. Muscle samples were obtained 1, 3, 6, 12, and 24 h after LPS injection in rats. Only the p67phox protein level after 24 h of LPS injection was significantly higher than control values, whereas the protein levels of other subunits remained similar to control values. (B) Left: Effects of 18 h of sepsis on basal O2- production in the diaphragm. **p < 0.01 compared with control values. Right: The influence of L-NAME on NADH-induced O2- production by the diaphragm of control and septic rats (18 h post-LPS). *p < 0.05 compared with NADH alone.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The main findings of this study are that (1) a nonmitochondrial NAD(P)H oxidase enzyme complex exists in human and rat skeletal muscle fibers; (2) this enzyme complex is localized in close proximity to the sarcolemma and consists of at least four subunits (p22phox, gp91phox, p47phox, and p67phox); (3) muscle NAD(P)H oxidase prefers NADH as a substrate and contributes in large part to basal O2- production in normal muscle fibers; (4) sepsis is associated with increased muscle O2- production but little change in muscle NAD(P)H oxidase expression; and finally, (5) NO synthesis inside skeletal muscle fibers exerts an inhibitory effect on muscle O2- levels in sepsis, but not under normal conditions.

Methodological Considerations

The use of LDCL as an accurate method to measure O2- radicals in biological systems has been challenged on the basis that redox recycling of lucigenin itself may actually produce O2- radicals (19). We believe that redox recycling of lucigenin is not a major component of LDCL signals observed in our study for the following reasons: (1) The accuracy of LDCL in measuring O2- radicals generated by several enzyme systems has been confirmed, using cytochrome c reduction and electron spin resonance (EPR) (20). We also found a linear relationship between LDCL and cytochrome c reduction in the presence of xanthine-xanthine oxidase (Figure 1); (2) production of O2- by vascular NAD(P)H oxidases remained largely unchanged when the lucigenin concentration was elevated from 5 to 250 µM (21); (3) evaluation of O2- production by vascular NAD(P)H oxidases, using 2-methyl-6-phenyl-3,7-dihydroimidazol[1,2-alpha ]pyrazine-3-one (CLA), which does not undergo redox recycling, yielded values similar to those measured with LDCL (22); and (4 ) our evaluation of muscle NAD(P)H oxidase activity was based on two methods, LDCL and NADH consumption measurements. Furthermore, our proposal that NAD(P)H oxidase exists in skeletal muscle fibers is supported by the observation that DPI exerts similar qualitative effects on both LDCL and NADH consumption measurements (Figures 2 and 5).

NAD(P)H Oxidase in Nonphagocytes

NAD(P)H oxidases are membrane-associated enzymes, which catalyze the one-electron reduction of molecular O2 using either NADH or NADPH as electron donors. Many structural and functional differences exist between phagocyte and nonphagocyte NAD(P)H oxidases including differences in orientation of the enzyme system, direction of O2- production, the rate and time course of O2- production, and subunit composition (7). In phagocytes, NADPH oxidase consists of five components: a plasma membrane-spanning cytochrome b558 (p22phox and gp91phox) and three cytosolic components (p47phox, p67phox, and p40phox), which associate with the cytochrome b558 on activation of phagocytes. Other proteins, including Rac2 and Rap1A, may also be involved in the assembly of phagocyte NADPH oxidase (6). The assembled oxidase complex in phagocytes spans the membrane and utilizes intracellular NADPH or NADH to transfer electrons to extracellular O2, resulting in extracellular release of O2- radicals. In nonphagocytes, mRNA of p22phox, gp91phox, p47phox, and p67phox subunits was found in endothelial and adventitial cells, whereas only p22phox and p47phox, not gp91phox, proteins were detected in vascular smooth muscles and mesangial cells (for review, see Reference 7). It has also been established that an NAD(P)H oxidase enzyme is responsible for extracellular release of O2- radicals in endothelial cells and fibroblasts, whereas intracellular O2- production has been attributed to NAD(P)H oxidase activity of vascular smooth muscles (23, 24).

We detected p22phox, gp91phox, p67phox, and p47phox mRNA and proteins in skeletal muscle fibers, whereas p40phox protein was localized in the blood vessels (Figure 8). Our study also indicates that, unlike phagocyte NADPH oxidase, all of the four subunits of muscle-specific NAD(P)H oxidase are constitutively associated with cell membranes, because none of these subunits were detected in the cytosolic fraction. The notion of a membrane-associated muscle-specific NAD(P)H oxidase is also confirmed by histochemical analysis using various antibodies (Figure 8) and by the observation that the muscle membrane fraction, rather than the cytosolic fraction, is largely responsible for NADH consumption. To our knowledge, our study is the first to document the presence of both mRNA and proteins of the four subunits of NAD(P)H oxidase in nonphagocytes. Of interest, we found that the muscle-specific p22phox subunit is larger (30 kDa) than that of phagocyte NADPH oxidase (22 kDa), an observation that could not be explained by species differences because anti-p22phox antibodies detected a 22-kDa protein in rat neutrophils (results are not shown). This observation and the finding that a 30-kDa p22phox subunit is present in sheep microglial cells (25) suggest that specialized forms of NAD(P)H oxidase proteins may be found in various nonphagocyte tissues.

We attempted to evaluate the contribution of NAPDH oxidase to ROS production in skeletal muscle fibers by measuring both the rate of production of O2- radicals and the rate of NADH consumption in the absence and presence of inhibitors of various ROS-producing enzymes. The fact that DPI and apocynin, but not rotenone, L-NAME, or indomethacin, inhibited LDCL signals and NADH consumption confirms that NAD(P)H oxidase, rather than mitochondrial enzymes, NOS, or cyclo-oxygenase, is largely responsible for O2- production and NADH use by the muscle membrane fraction. Moreover, the observation that NADH-induced O2- production was more effectively inhibited by cell membrane-permeable Tiron than cell membrane-impermeable SOD (Figure 2C) suggests that a significant proportion of O2- radicals synthesized by muscle NAD(P)H oxidase resides intracellularly. We should emphasize in this respect that lucigenin is capable of passing through cell membranes and can be used to detect intracellular O2- production (26).

Production of O2- Radicals in Sepsis

It is well established that sepsis causes oxidative stress in skeletal muscle fibers as a result of both an increase in ROS production and a significant decline in the antioxidant capacity of these muscles (27). Our results indicate that basal O2- radical production in the diaphragm of septic rats was significantly higher than that of control animals, confirming that ROS production rises in septic muscles. The molecular sources responsible for the sepsis-induced rise in ROS production remain under investigation. In the vascular system, sepsis or proinflammatory cytokines elicit upregulation of p22phox, gp91phox, and p67phox subunit expression (30, 31). It has also been reported that O2- production by vascular smooth muscle NAD(P)H oxidase increases significantly within minutes of exposure to tumor necrosis factor alpha  (TNF-alpha ), suggesting that augmentation of NAD(P)H expression might not be necessary for increased activity of this enzyme (31). We speculate that NADPH oxidase contributes significantly to the rise in diaphragmatic O2- production in septic rats and that increased activity of this enzyme in septic muscles is mediated through multiple mechanisms including upregulation of subunit expression, especially that of the p67phox subunit, as well as posttranslational modifications involving subunit assembly and phosphorylation of specific subunits. Clearly, more research is needed to elucidate the mechanisms through which sepsis influences NAD(P)H oxidase activation.

Our finding of a significant elevation of NADH-stimulated O2- radical production in septic muscles when NOS activity was inhibited suggests that the production of NO can modulate muscle O2- radical production. In normal muscle fibers, NO is synthesized mainly by the constitutive NOS isoforms (8, 9). However, in septic animals, larger quantities of NO are synthesized inside skeletal muscle fibers by the inducible (iNOS) isoform of NOS (32). It is, therefore, likely that induction of iNOS expression and enhanced NO production in septic muscles might have masked the increase in O2- production in these muscles. There are two main mechanisms through which NO influences muscle NAD(P)H oxidase activity: (1) NO can directly react with O2- in a diffusion-limited reaction to produce peroxynitrite (33). Although earlier reports suggest that peroxynitrite formation is limited to pathological conditions such as severe sepsis (33), more recent studies indicate the reaction between NO originating from endothelial cells and O2- generated by nonphagocyte NAD(P)H oxidase in adventitial cells occurs even in normal blood vessels (34); and (2) NO is capable of inhibiting the assembly of cytosolic and membrane-bound subunits of phagocyte NADPH oxidase radicals (11). Whether the assembly of muscle NAD(P)H oxidase is also inhibited by NO remains to be determined. We speculate that this inhibitory effect of NO on O2- levels in septic muscle could be partly responsible for the protective roles of NOS activity in attenuating sepsis-induced ventilatory muscle contractile dysfunction (35).

In summary, our study indicates the presence of an NAD(P)H oxidase enzyme complex inside skeletal muscle fiber, which consists of at least four subunits (p22phox, gp91phox, p67phox, and p47phox) and has characteristics that are similar to those of nonphagocyte NAD(P)H oxidases in other tissues. This NAD(P)H oxidase enzyme complex is constitutively active and contributes significantly to basal ROS production inside skeletal muscle fibers under normal conditions and in response to sepsis.

    Footnotes

Correspondence and requests for reprints should be addressed to S. Hussain, M.D., Room L3.05, Critical Care Division, Royal Victoria Hospital, 687 Pine Ave. West, Montreal, Quebec, Canada H3A 1A1. E-mail: sabah.hussain{at}muhc.mcgill.ca

(Received in original form March 8, 2001 and accepted in revised form October 18, 2001).

Funded by grants from the Canadian Institute of Health Research and National Institutes of Health (R01 HL66757 and AR42426).
This article has an online data supplement, which is accessible from this issue's table of contents online at www.atsjournals.org

Acknowledgments: The authors are grateful to Ms. J. Nicolac and Mr. L. Franchi for technical expertise and to Ms. C. Mutter for editorial expertise.
    References
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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