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Am J Physiol Lung Cell Mol Physiol 292: L1013-L1022, 2007. First published December 22, 2006; doi:10.1152/ajplung.00112.2006
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Nitric oxide production in the ventilatory muscles in response to acute resistive loading

Theodoros Vassilakopoulos,1 Karuthapillai Govindaraju,2 Dimitrios Parthenis,1 David H. Eidelman,2 Yasu Watanabe,3 and Sabah N. A. Hussain2

1Department of Critical Care and Pulmonary Services, University of Athens Medical School, Evangelismos Hospital, Athens, Greece; 2Critical Care and Respiratory Divisions and Meakins-Christie Laboratories, Department of Medicine, McGill University Health Centre, Montreal, Québec, Canada; and 3Department of Cell Physiology, Faculty of Medicine, Kagawa University, Kagawa, Japan

Submitted 26 March 2006 ; accepted in final form 20 December 2006


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The effect of muscle activation on muscle nitric oxide (NO) production remains controversial. Whereas NO release increases in in vitro activated muscles and in vivo limb muscles, diaphragmatic NO synthase (NOS) activity declines after 3 h of inspiratory resistive loading (IRL). We tested in this study the hypotheses that acute IRL decreases diaphragmatic NO derivatives levels and reduces protein expression of neuronal (nNOS), endothelial (eNOS), and inducible (iNOS) NO synthases, as well as 3-nitrotyrosine formation. Anesthetized, tracheostomized, spontaneously breathing adult rats were subjected to IRL (50% of the maximum inspiratory pressure) for 1, 3, or 6 h. Quietly breathing rats served as controls. After 3 h of IRL, muscle eNOS and nNOS protein levels rose by 80 and 60% of control values, respectively. Whereas eNOS expression did not change any further, nNOS expression reached 550% of control values after 6 h of IRL. Strong iNOS protein expression was detected in the diaphragms after 6 h of IRL. Total NO derivatives levels in the diaphragm declined during IRL as a result of reduction in nitrate, nitrite, and nitrosothiols. Diaphragmatic protein tyrosine nitration decreased in response to IRL, and this reduction was mainly due to reduced tyrosine nitration of enolase and aldolase. We conclude that diaphragmatic NO derivatives levels decline in response to IRL and that the rise in diaphragmatic NOS protein expression may be a compensatory response designed to counterbalance the decline in NOS activity.

nitrite; muscle contraction; diaphragm; nitric oxide synthases; nitrosothiols

NITRIC OXIDE (NO) is synthesized inside skeletal muscle fibers by the endothelial (eNOS) and neuronal (nNOS) NO synthases (26, 27, 42). The eNOS isoform is localized in the mitochondria (27) and in sarcolemmal caveolae (at least in cardiac myocytes) associated with caveolin-3 (14). The nNOS isoform directly associates with the dystrophin complex and is localized in close proximity to the sarcolemma of mainly type II fibers (11). Although very low levels of the inducible NOS (iNOS) isoform may be expressed in skeletal muscles of normal mammals, the expression of this isoform in limb and ventilatory muscles rises significantly and in a transient fashion in response to proinflammatory conditions such as severe sepsis in humans (29) and bacterial endotoxemia in rodents (20). NO can promote many important processes inside skeletal muscle fibers such as Ca2+ release from the sarcoplasmic reticulum, glucose metabolism, and blood flow (42). However, excessive NO production primarily by the iNOS isoform may have deleterious effects on muscle contractile performance (8) and sarcolemmal integrity (32).

Resistive breathing is a clinically relevant form of acute exercise for the respiratory muscles, since it is encountered in various disease states such as upper airway obstruction, snoring, or acute asthma attacks, and exacerbations of chronic obstructive pulmonary disease. Despite the importance of NO in the regulation of muscle function, little information is available regarding the effect of strenuous diaphragmatic contraction on muscle NO levels. Studies assessing the effects of increased diaphragm activation on NO production produced rather conflicting results. Very short term inspiratory resistive loading (IRL) (<10 min) did not change NOS activity (7), whereas 3 h of IRL resulted in a decline in ventilatory muscle NOS activity with no change in NOS protein expression (15). In contrast, long term increases in diaphragmatic activity elicited by the hyperventilation that accompanies chronic exercise training resulted in upregulation of both NOS activity and eNOS and nNOS protein expressions (46). IRL results in the production of proinflammatory cytokines within the working diaphragm in a time-dependent manner (47). Exposure of striated muscles to proinflammatory cytokines induces muscle iNOS expression (48). Thus we hypothesized that the regulation of NO levels and NOS protein expression inside the diaphragm secondary to IRL is time-dependent. Furthermore, since exercise leads to phosphorylation of nNOS inside skeletal muscles by an AMP-activated protein kinase (12), and phosphorylated NOS exhibits reduced activity (28), we also hypothesized that increases of nNOS phosphorylation inside the diaphragm reflect reduced NO derivatives secondary to IRL.

IRL is also accompanied by enhanced production of reactive oxygen species inside diaphragmatic muscle fibers (2), which, when combined with NO production, leads to the formation of peroxynitrite, which in turn targets proteins and lipids and leads to posttranslational modifications and inactivation of various enzymes including those involved in energy production, fatty acid metabolism, and the defense against oxidative stress (6). An important posttranslational protein modification elicited by peroxynitrite is nitration of tyrosine residues, which can elicit enzyme dysfunction, particularly when the tyrosine residues targeted by peroxynitrite are critical for protein function (6). Several recent reports, including ours (5), have documented a significant increase in protein tyrosine nitration in the diaphragm during the course of sepsis or endotoxemia (9). We thus hypothesized that the acute increase in ventilatory muscle activation as a result of IRL would alter protein tyrosine nitration in the ventilatory muscles. In addition, the identity of proteins inside the ventilatory muscles, which are tyrosine-nitrated by endogenous peroxynitrite and NO production, remains unknown.

The two main aims of this study were: 1) to assess the influence of IRL on NO derivatives levels and NOS protein isoform expression in the diaphragm; and 2) to identify the nature of tyrosine-nitrated proteins inside the diaphragm and whether the level of diaphragm protein tyrosine nitration (footprints of peroxynitrite formation) is influenced by IRL.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. Reagents for protein measurement were purchased from Bio-Rad (Hercules, CA). Gels and loading buffers for immunoblotting were obtained from Novex (San Diego, CA). Chemicals were purchased from Sigma Chemical (St. Louis, MO). Primary monoclonal anti-eNOS (cat. no. 610297), anti-nNOS (cat. no. 611852), and anti-iNOS (cat. no. 610432) antibodies were obtained from Transduction Laboratories (Lexington, KY). For detection of nNOS phosphorylation at Ser847, we used a polyclonal antibody (NP847) (19). Monoclonal anti-3-nitrotyrosine was obtained from Cayman Chemical (cat. no. 189542; Ann Arbor, MI). Secondary antibodies for immunoblotting were obtained from Transduction Laboratories. Reagents for enhanced chemiluminescence (ECL) detection were obtained from Chemicon (Temecula, CA).

Animal preparation. The Animal Research Committee of McGill University approved the procedures used in this study, which were carried out according to the guiding principles for the care and use of animals based on the Helsinki Declaration. Pathogen-free, adult male Sprague-Dawley rats (300–325 g; Charles River Laboratories) were studied. Animals were lightly anesthetized with pentobarbital sodium (30 mg/kg ip) and were given supplemental doses (10 mg/kg) as needed (on the basis of the absence or presence of corneal reflexes). The animals were tracheostomized with polyethylene tubing (internal diameter 2.2 mm; Intramedic, Clay Adams), which was sutured firmly in place with a silk tie. The cannula was connected to a two-way, nonrebreathing valve (model 2300; Hans Rudolph, Kansas City, MO). Tracheal pressure was measured with a differential pressure transducer (Validyne, Northridge, CA) connected to the tracheal cannula via a side port. The inspiratory line was connected to a 5-l bag (by means of needles of various internal diameter, see below), which was continuously filled with 100% O2. Preliminary experiments indicated that this gas composition prevents the development of hypoxemia during resistive loading. An arterial catheter (22-gauge) was placed into the internal carotid artery for sampling of arterial blood. Arterial blood gases were analyzed by means of an automatic blood-gas analyzer (AVL model 995; Instrumentation Laboratories, Lexington, MA). A catheter, placed into the jugular vein, was used to access the venous circulation. At the end of the surgical procedure, a 30-min stabilization period was allowed. The animals (n = 8 in each group) were then randomly assigned to a period of 1, 3, or 6 h of IRL. Animals undergoing the same surgical preparation but breathing against no load for 1, 3, and 6 h served as controls. In each animal, maximum tracheal pressure was measured before IRL by occluding the inspiratory port of the Hans Rudolph valve for a period of 30 s. The degree of resistance was set initially to result in the generation of tracheal pressures of 50% of maximum tracheal pressure, respectively, using needles of appropriately sized internal diameter as resistors connected to the oxygen containing bag. At the end of the period of IRL, the inspiratory resistance was removed, and maximum tracheal pressure was remeasured. Results from these experiments concerning other research questions have already been reported (47). The animals were euthanized with an overdose of pentobarbital sodium. The chest and the abdomen were opened, and the diaphragm was quickly excised and frozen in liquid nitrogen.

Measurement of NO derivatives: nitrate, nitrite, and nitrosothiols. The chemiluminescence assay for the measurement of nitrate (NO3–), nitrite (NO2–), and nitrosothiol (RSNO) in the diaphragmatic homogenate was carried out using a NO analyzer (NOA 280i; Sievers Instrument, Boulder, CO) interfaced through an analog-to-digital converter board to a personal computer.

Total NO. The assay utilizes the total reduction of nitrate, nitrite, and nitrosothiol to NO under acidic pH condition in the presence of vanadium chloride (VCl3) and potassium iodide (KI). In brief, cell lysates were diluted with NO-free HPLC-grade water (Fisher Scientific, Fair Lawn, NJ). Thirty microliters of the diluted sample was injected using a Hamilton gas-tight microsyringe through the rubber septa into a glass purging vessel containing saturated VCl3 (in 1 N HCl) and 1% KI in glacial acetic acid solution at 90°C, purged continuously with helium gas. Under this condition, NO was released immediately (Eqs. 1–3) from the samples and passed on to NO analyzer through ice-cold trap containing 1 N NaOH and 100 mM cysteine. The trap was added to prevent contamination of the analyzer with acid or iodine vapor. In the analyzer, NO was reacted with ozone-forming excited state NO, which emits light, and captured by photomultiplier tube (PMT). The amount of NO was then quantified by analysis, and integration of digitally recorded signal from the PMT tube using data acquisition and analysis software from Sievers Instrument NO analyzer was calibrated with standard nitrate solutions (0.8–50 µM). Duplicate or triplicate analyses were carried out for each sample. RSH is defined as amino acid (R) with a sulfhydryl group (SH).

Formula 1(1)

Formula 2(2)

Formula 3(3)

Formula 4(4)

Formula 5(5)

NO2– measurement. The chemiluminescence assay for the measurement of nitrite involves the reduction of nitrite to NO under acidic condition, using glacial acidic acid, in the presence of reducing agent KI (1%). At acidic pH nitrous acid, HNO2 is formed and then reduced to NO (Eq. 2). The release of NO from nitrite was measured as described above and was quantified by calibrating with standard nitrite solutions (0.4–25 µM). This assay is not affected by the presence of nitrate and nitrosothiol.

RSNO measurement. Nitrosothiol measurement was done based on the Saville method that involves mercury ion-mediated heterolytic cleavage of the S-NO bond and the release of nitrosonium ion (NO+). In the presence of iodide, NO+ is reduced to NO. In brief, the assay involves the reduction of both nitrosothiol and nitrite to NO under acidic condition, using glacial acetic acid, in the presence of mercuric chloride, HgCl2 (4 mM), and KI (1%). Nitrate is not affected by these reactions. RS is defined as thiols.

Formula 6(6)

Formula 7(7)
The release of NO from both nitrosothiol and nitrite was measured as described above and was quantified by calibrating with standard S-nitrosoglutathione (GSNO) solutions (0.4–25 µM). The amount of nitrosothiol was calculated by subtracting nitrite NO value from nitrite and nitrosothiol NO value.

NO3– measurement. Nitrate levels were calculated by subtracting both nitrite and nitrosothiol NO value from total NO (NOx) values obtained by vanadium reduction assay.

Immunoblotting. Frozen muscle samples were homogenized in a buffer (HEPES 20 mM, pH 7.4, 0.2 mM PMSF, 1 µM leupeptin, 1 µM pepstatin A, 0.4 mM EDTA, 0.2 mM sodium orthovanadate), centrifuged at 1,000 g for 10 min, and supernatants (crude muscle homogenates, 80 µg total protein per sample) were then separated onto Tris-glycine SDS-PAGE. Proteins were then transferred to polyvinylidene difluoride (PVDF) membranes and probed overnight at 4°C with primary antibodies. The eNOS, nNOS, iNOS, and phospho-nNOS (NP847) proteins were detected with selective monoclonal antibodies (1:500 for each antibody) with lysates of endothelial cells, pituitary cells, and cytokine-activated macrophages as positive controls. The formation of 3-nitrotyrosine was detected with a monoclonal antibody as previously indicated (5). Specific proteins were detected with horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody and an ECL kit and quantified with Image-Pro Plus software (Media Cybernetics). For nitrated proteins, total 3-nitrotyrosine optical density (OD) was calculated for each sample by adding the OD of the individual positive protein bands. Specificity of anti-3-nitrotyrosine antibody was evaluated by preincubation of each primary antibody with either 10 mM nitrotyrosine or 10-fold excess of peroxynitrite tyrosine-nitrated bovine serum albumin (generously provided by Dr. H. Ischiropoulos, University of Pennsylvania).

Myeloperoxidase activity. Crude muscle homogenates were mixed with 50 mM potassium phosphate buffer (pH 6.0) containing o-dianisidine dihydrochloride and H2O2 (35). Absorbance was measured at 460 nm for 60 min. MPO activity was calculated in units: change in absorbance per minute per gram of protein.

Identification of tyrosine-nitrated proteins using 2D electrophoresis. Crude homogenates of diaphragms obtained from quietly breathing rats (400 µg of protein per sample) were added to ice-cold trichloroacetic acid (TCA; 15% final concentration). The samples were then incubated for 10 min on ice, centrifuged for 10 min at 14,000 g, and the pellets were then washed three times with ethanol ethyl acetate and centrifuged at 14,000 g for 15 min. The pellets were resuspended in two-dimensional (2D) rehydration buffer (8 M urea, 4% [(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate [CHAPS], 0.2% ampholytes [pH 3–10], and 50 mM DTT). Each muscle sample was then separated into two portions (100 µg total each) and both portions underwent 2D electrophoresis. First-dimensional protein separation was performed with Protean IEF Cell (Bio-Rad). Samples were applied to immobilized pH gradient (IPG) strips (17-cm nonlinear pH 3–10, Bio-Rad) for 1 h at room temperature. The strips were then covered with mineral oil overnight and isoelectric focusing was performed at 10,000 V/h for a total of up to 60–100 kV/h. For the second dimension, the IPG strips were equilibrated in room temperature for 10 min in equilibration buffer (6 M urea, 2% SDS, 0.05 mM Tris·HCl, 20% glycerol), to which 2% DTT was added before use. An additional 10-min equilibration period was then used with equilibration buffer, to which 2.5% iodoacetamide was added. The strips then were embedded in 0.7% agarose on the top of 10% acrylamide slab gels (23.5 x 18 x 0.15 cm) containing a 4% stacking gel. The second dimension SDS-PAGE was performed for 5 h, 30 mA per gel at 300 V. One of the resulting 2D gels for each muscle sample was then stained with silver stain. Gels were fixed overnight in a fixation solution (10% acetic acid, 45% methanol), and then rinsed twice in water, sensitized for 1 min in 0.02% sodium thiosulfate followed by rinsing in water and immersion for 1 h in a silver nitrate solution (0.5 mM silver nitrate, 0.026% formaldehyde). Gels were then rinsed twice in water and developed in a developer solution (0.1 M sodium carbonate, 0.01% formaldehyde, 0.00125% sodium thiosulfate). A stop solution (0.1 M Tris, 2% acetic acid) was then added for 30 min followed by rinsing with water for 5 min. Gels were then stored in 2% acetic acid. The second gel derived from a given sample underwent electrophoretical transfer to PVDF membrane and immunoblotting with a monoclonal anti-3-nitrotyrosine antibody. Gels and PVDF membranes were imaged with a digital camera and aligned (Image-Pro Plus) so as to identify positive carbonylated protein spots on the gels.

Mass spectrometry. Tyrosine-nitrated protein spots were cut out of the gels and taken for in-gel digestion on a robotic MassPrep workstation (Micromass, Waters Milford, MA). In brief, the gel pieces were destained, reduced with 10 mM dithiothreitol, alkylated with 55 mM iodoacetamide, and incubated with 6 ng/µl trypsin for 5 h at 37°C. Peptides were then extracted with 1% formic acid/2% acetonitrile. Identification of the digested proteins was completed using a liquid chromatography quadruple time-of-flight (LC-Q-Tof) Mass Spectrometer (MicroMass). The digests were loaded into 10-cm capillary PicoFrit column filled with C18 stationary phase and eluted by linear gradient of 5–70% acetonitrile in 0.1% formic acid at the flow rate 200 µl/min. The eluted peptides were electrosprayed into Q-Tof, and the precursor ions were selected and subjected to fragmentation by collision with argon (MS/MS). The MS/MS data were submitted to Mascot (Matrix Science, London, United Kingdom) for a search against the National Center for Biotechnology Information (NCBI) nonredundant database.

To confirm the time course of changes in tyrosine nitration of specific proteins, we first detected tyrosine-nitrated proteins in crude diaphragm lysates using one-dimensional (1D) electrophoresis as described above. The primary and secondary antibodies were then stripped from the PVDF membranes by incubation with 0.2 N NaOH solution for 5 to 60 min. Following several washes, the membranes were then probed with antibodies selective to enolase, aldolase (Santa Cruz Biotechnology), and carbonic anhydrase III (CAIII). Specific proteins were then detected with HRP-conjugated secondary antibodies and ECL kit. Tyrosine nitration and specific protein blots were then scanned with an imaging densitometer. The ODs of tyrosine-nitrated proteins and specific proteins were quantified using Image-Pro Plus.

RNA extraction. Total RNA was isolated using the RNeasy Midi Total RNA Isolation System kit (Qiagen) with proteinase K and DNaseI treatments. First-strand cDNA was synthesized using SuperScript II reverse transcriptase (Invitrogen) with random hexamers, according to the manufacturer's instructions, in a total volume of 20 µl. Samples were heated for 5 min at 70°C, cooled on ice, and then the RT buffer (containing 5x first-strand buffer, DTT, and RNase inhibitor) was added, and samples were incubated for 2 min at room temperature. One microliter of SuperScript enzyme was then added, and samples were then incubated for 10 min at room temperature, 50 min at 42°C, and finally 5 min at 90°C.

Real-Time PCR. Expression of prepro-endothelin-1 (prepro-ET-1), prepro-ET-3, and protein inhibitor of nNOS (PIN) mRNA in diaphragm samples in the resistive loading groups relative to that of quiet breathing rats was investigated using real-time quantitative RT-PCR based on SYBR Green fluorescence methodology. Gene specific primers were designed for each gene. Rat GAPDH was used as the endogenous control gene. The primers were as follows: prepro-ET-1, forward 5'-GCTCCTGCTCCTCCTTGATG-3', reverse 5'-CTCGCTCTATGTAAGTCATGG-3'; prepro-ET-3, forward 5'-GCACTTGCTTCACTTATAAGG-3', reverse 5'-CAGAAGCAAGAAGCATCAGTTG-3'; PIN, forward 5'-ATGTGCGACCGAAAGGCCGTAGATC-3', reverse 5'-TTAACCAGATTTGAACAGAAGAATGGCC-3'; GAPDH, forward 5'-AAGAAGGTGGTGAAGCAGGCG-3', reverse 5'-ACCAGGAATGAGCTTGACAA-3'. Reactions were performed with SYBR Green Universal PCR Master Mix (Qiagen) in a 50-µl reaction volume. All reactions were performed in duplicate and included a negative control. PCR reactions were performed in the model 7500 real-time PCR System (Applied BioSystems). Cycling conditions were 15 min at 95°C, 40 cycles of 15 s at 95°C, 30 s at 57°C, and 34 s at 72°C. Relative quantification of the mRNA levels of the target genes was determined using the threshold cycle ({Delta}{Delta}Ct) method. Briefly, the amount of target (prepro-ET-1, prepro-ET-3, and PIN) was normalized to the endogenous reference gene (GAPDH), and its expression in the diaphragm of IRL animals was expressed as percentage of those measured in quiet breathing animals.

Statistical analysis. Values reported are means ± SE. Since some of the data were not normally distributed, comparisons were made using Kruskal-Wallis one-way analysis of variance (ANOVA) followed by Wilcoxon rank sum test for post hoc comparisons. A P value of 0.05 was initially considered as statistically significant, and was accordingly adjusted using a Bonferroni-type procedure for multiple comparisons.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Maximum peak tracheal pressure measured before IRL averaged 70 ± 1 cmH2O (n = 24). Peak inspiratory tracheal airway pressure developed by the animals during IRL averaged 35 ± 3 cmH2O (50 ± 8% of maximum peak tracheal pressure). Table 1 lists arterial blood gases in the four groups of animals before and after quiet breathing or IRL. Whereas arterial blood gases remained unchanged after the experimental period in the quietly breathing animals, IRL for 1, 3, and 6 h resulted in a significant decline in arterial pH, PO2, and a significant rise in arterial PCO2 (Table 1).


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  		Table 1. Arterial blood gases measured in the four groups of animals

 
NO production and NOS protein expression. IRL resulted in significant decline in the level of NOx (P < 0.05), nitrate (P < 0.05), nitrite (P < 0.05), and nitrosothiols (P < 0.05), which was already evident after 1 h of IRL and remained depressed thereafter (Fig. 1). Unlike this decline in NO derivatives levels, IRL elicited a significant upregulation in the protein expression of the three NOS isoforms in the diaphragm in a time-dependent fashion (Fig. 2A). After 3 h of IRL, nNOS protein levels increased by 60% (P < 0.05) and eNOS by 80% (P < 0.05) compared with quietly breathing animals, whereas iNOS protein levels did not change (Fig. 2B). When IRL was prolonged to 6 h, eNOS protein levels did not exhibit any further change, but nNOS protein expression rose by 500% (P < 0.01) and iNOS protein levels increased for the first time to 400% of that of quietly breathing animals (P < 0.01) (Fig. 2, A and B). Phosphorylation of nNOS protein at Ser847 was detectable in diaphragm lysates obtained from quietly breathing rats (average OD of 24.9 ± 4.1 arbitrary units [a.u.]; Fig. 2C). The intensity of nNOS phosphorylation in the diaphragm at this residue was not influenced by IRL (OD of 25.3 ± 6.4 a.u. after 6 h of IRL.; Fig. 2C).


Figure 1
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  		Fig. 1. Total nitric oxide (NO) (NOx), nitrate, nitrite, and nitrosothiols (normalized per milligram of total protein) in diaphragm homogenates obtained from quietly breathing rats (QB) and rats exposed to 1, 3, and 6 h of inspiratory resistive loading (IRL). *P < 0.05 compared with that of quietly breathing animals. Results represents means ± SE.

 

Figure 2
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  		Fig. 2. A: immunoblotting of diaphragm homogenates with antibodies selective to neuronal (nNOS), endothelial (eNOS), and inducible (iNOS) NO synthases and tubulin. Note the increase in eNOS and nNOS protein expression and the induction of iNOS protein during IRL. QB, quiet breathing animals maintained for 1 h; +ve, positive control. No differences were found in terms of NOS expression between quietly breathing animals maintained for 1, 3, and 6 h. B: means ± SE of diaphragm NOS protein optical densities (normalized as percentage of those obtained from diaphragms of animals, which were quietly breathing). *P < 0.05 compared with that of quietly breathing animals. C: immunoblotting of diaphragm homogenates with antibodies selective to total nNOS and phosphorylated nNOS at Ser847. Note that the intensity of nNOS phosphorylation at this residue did not change during IRL compared with that observed in the diaphragm of quietly breathing animals. OD, optical density.

 
MPO activity. IRL elicited no change in the MPO activity in the diaphragms, which averaged 77.8 ± 7.1 units in animals breathing against no load, 85.6 ± 18.6, 87.73 ± 19.3, and 82.7 ± 12.6 units after 1, 3, and 6 h of IRL, respectively (P = not significant).

Expression of endothelins and PIN. Within 1 h of IRL, prepro-ET-1 mRNA expression in the diaphragm rose by more than 1,000% compared with that of quietly breathing rats (P < 0.05). After 3 and 6 h of IRL, prepro-ET-1 mRNA remained elevated, whereas that of prepro-ET-3 increased significantly only after 3 h of IRL. No significant alterations in PIN mRNA were observed in response to IRL (Fig. 3).


Figure 3
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  		Fig. 3. The intensity of mRNA expression of prepro-endothelin-1 (prepro-ET-1), prepro-ET-3, and protein inhibitor of nNOS (PIN) in diaphragm samples obtained from rats exposed to 1, 3, and 6 h of IRL. Results are shown as percentage changes from those obtained in quietly breathing animals. *P < 0.05 compared with that of quietly breathing animals.

 
Protein tyrosine nitration. Anti-3-nitrotyrosine antibody detected several positive tyrosine-nitrated protein bands of 82, 50, 44, 41, 39, 35, and 32 kDa in diaphragm samples of quietly breathing rats (Fig. 4A). IRL resulted in a significant decrease in the level of total nitrotyrosine from 188.7 ± 22.1 a.u. in quietly breathing animals to 127.1 ± 10.5 and 104.3 ± 9.5 a.u. after 3 and 6 h, respectively (P < 0.037). This reduction in total protein tyrosine nitration inside the diaphragm in response to IRL was primarily due to reduction in the tyrosine nitration intensity of the 50-kDa and 44-kDa protein bands (Fig. 4B). To identity tyrosine-nitrated proteins inside the diaphragms, we performed 2D electrophoresis and immunoblotting with anti-3-nityrosine antibody using diaphragm lysates obtained from quietly breathing rats. Figure 5 illustrates a representative example of a 2D protein map of crude diaphragm homogenates of a quietly breathing rat in which tyrosine-nitrated proteins were detected using anti-3-nitrotyrosine antibody (bottom) followed by excision of positive tyrosine-nitrated proteins spots from silver-stained gels (top) and mass spectrometry analysis (Table 2). Ten positive tyrosine-nitrated proteins were detected with varying intensities (Fig. 5). Three tyrosine-nitrated protein spots with an apparent molecular mass of ~85 kDa were identified to be myosin heavy chain (spots 1–3; Fig. 5 and Table 2). In addition, three tyrosine-nitrated proteins with apparent molecular masses of 50, 44, and 40 kDa were identified to be the glycolysis enzymes enolase 3beta, aldolase, and GAPDH (spots 4–8). Finally, two strongly tyrosine-nitrated protein spots with an apparent molecular mass of 32 kDa (spots 9 and 10) were identified to be CAIII (Fig. 5 and Table 2). To verify the identity of these tyrosine-nitrated proteins and to monitor tyrosine nitration intensity of these proteins during IRL, we performed 1D electrophoresis and immunoblotting with 3-nitrotyrosine antibody. This antibody was then stripped, and membranes were reprobed with antibodies selective to aldolase, enolase, or CAIII. Figure 6A shows that the 50- and 44-kDa tyrosine-nitrated proteins inside the diaphragm were indeed enolase and aldolase, respectively, and that the intensity of tyrosine nitration of these two proteins but not their total levels declined significantly in response to IRL. By comparison, the 32-kDa tyrosine-nitrated protein was verified to be CAIII and neither the intensity of tyrosine nitration nor the level of total CAIII protein inside the diaphragm changed significantly after 6 h of IRL (Fig. 6B).


Figure 4
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  		Fig. 4. A: a representative example of anti-3-nitrotyrosine immunoblot of diaphragm homogenates obtained from quietly breathing rats (maintained for 1 h) and rats exposed to 3 and 6 h of IRL. No differences in protein tyrosine nitration were observed between animals quietly breathing for 1, 3, and 6 h. B: means ± SE of tyrosine nitration intensity of specific proteins (expressed in arbitrary units) measured in diaphragm homogenates obtained from quietly breathing rats and rats exposed to 3 and 6 h of IRL. *P < 0.05 compared with that of quietly breathing animals. Note the significant decline in tyrosine nitration intensity of proteins with apparent molecular masses of 50 and 44 kDa.

 

Figure 5
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  		Fig. 5. Detection of protein tyrosine nitration in crude diaphragm homogenates obtained from quietly breathing rats using 2-dimensional (2D) electrophoresis. Representative silver-stained 2D gel (top) and 2D anti-3-nitrotyrosine blot (bottom) are shown. Ten positively tyrosine-nitrated proteins were detected in the 2D blot, and corresponding protein spots are indicated by circles in the 2D gel. Identity of each tyrosine-nitrated protein spot is listed in Table 2.

 

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  		Table 2. Tyrosine-nitrated proteins in the cytosolic fraction of rat diaphragms

 

Figure 6
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  		Fig. 6. Confirmation of tyrosine nitration of aldolase, enolase, and carbonic anhydrase III (CAIII) proteins in the diaphragms of quietly breathing rats and rats exposed to 6 h of IRL. Immunoblotting with anti-3-nitrotyrosine antibody was performed first. The antibody was then stripped, and membranes were then probed with antibodies selective to aldolase and enolase (A) and CAIII (B). Note that the intensity of tyrosine nitration of aldolase and enolase declined in response to IRL but total protein levels of these 2 enzymes remained unchanged. Also note that the CAIII tyrosine nitration level did not change in response to IRL.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The main findings of this study are: 1) acute IRL resulted in significant decline in the diaphragmatic NO derivatives levels despite a significant increase in eNOS, nNOS, and iNOS protein expression; 2) the decline in diaphragmatic NO derivatives levels was not due to increased phosphorylation of nNOS protein isoform but was associated with increased ET-1 mRNA expression within the diaphragm; and 3) three glycolysis enzymes (aldolase, enolase, and GAPDH) and CAIII are tyrosine-nitrated in quietly breathing rat diaphragms. IRL elicited a significant decline in tyrosine nitration of aldolase and enolase.

NO production and muscle activation. Few studies have evaluated the influence of an acute increase in muscle activity on the rate of NO production by various striated muscles. Electrical stimulation of the extensor digitorum longus or the soleus increased muscle NO production (4, 45). NOS activity increased by ~40% in the gastrocnemius muscle after 45 min of treadmill running (40). Increased nitrate concentration was detected in peripheral skeletal muscles (vastus intermedius, gastrocnemius, plantaris) after various treadmill running protocols (38). In contrast, treadmill exercise (of similar duration and intensity) elicits a decrease in NOx levels in the heart (21), a response similar to that we (15) described earlier in the diaphragm during IRL. These results along with our current findings indicate that the influence of an acute increase in muscle activation on the levels of NO is different for peripheral (limb) skeletal muscles and for the heart and the ventilatory muscles.

We found that the levels of NOx and its stable end-products in the diaphragm decreased significantly in response to acute increases in diaphragm activation during IRL. These results suggest that NO production might have been attenuated during IRL and/or NO removal might be augmented. The precise mechanisms involved in this response remain unclear. There are several possible mechanisms through which muscle NOS activity might have been attenuated. First, the activity of various NOS isoforms is regulated by posttranslational modifications such as phosphorylation at specific residues, which leads to either inhibition of NOS activity as in the case of nNOS phosphorylation at Ser847 (28) or eNOS phosphorylation at Ser635, Ser1179, and Thr497, which induces an increase in eNOS activity (10, 33). We focused on measuring nNOS phosphorylation at Ser847 primarily because nNOS is the main source of NO production inside skeletal muscle fibers (42). Our results indicate no significant alterations in the intensity of diaphragmatic nNOS phosphorylation at Ser847 during IRL, thereby excluding this mechanism as a major cause of reduced muscle NO production. Other forms of nNOS posttranslational modifications or changes in diaphragmatic eNOS phosphorylation cannot be excluded in our experiments. Second, NOS activity is sensitive to changes in pH, PO2, and PCO2 (1). Since IRL elicited a significant increase in arterial PCO2, whereas arterial pH and PaO2 declined significantly in response to IRL (Table 1), one could argue that reduction in muscle NO levels in response to IRL might have been the results of alterations in arterial blood gases. However, none of our animals was hypoxic. Moreover, NO levels were significantly reduced after 1 h of IRL with no further change thereafter, whereas arterial pH continued to decline, and arterial PCO2 continued to rise after 3 and 6 h of IRL compared with 1 h of IRL (Table 1). These observations suggest that reduction in NO levels during IRL was not simply due to acidosis or hypercapnia, although the local intradiaphragmatic gas tension milieu is expected to be different from the systemic blood levels. It should also be acknowledged that the imposed hyperoxia (which was necessary for the prevention of hypoxia secondary to the development of hypercapnia induced by IRL) might have contributed to the observed changes through its effect on reactive oxygen species formation, although the level of hyperoxia was similar in quietly breathing and IRL rats. Third, a significant reduction in cofactor availability, especially that of tetrahydrobiopterin (BH4), in response to increased diaphragmatic activity would result in reduced NOS activity. Little is known about regulation of skeletal muscle BH4 production; however, it is known that serum levels of BH4 increase in response to whole body exercise (18). Furthermore, the expression of GTP cyclohydrolase I, the rate-limiting enzyme in BH4 synthesis, is induced along with iNOS by proinflammatory cytokines such as TNF-{alpha} and IL-1beta (41). Our (47) recent observation that the production of both of these two cytokines is elevated inside the diaphragm during IRL suggests that diaphragm GTP cyclohydrolase I expression might have been induced during IRL. Taken together, we propose that it is rather unlikely that diaphragm BH4 availability is reduced during IRL. Fourth, alterations in the levels of endogenous NOS inhibitors may lead to a reduction in diaphragmatic NOS activity. These inhibitors include endogenous asymmetrically methylated forms of arginine such as NG-monomethyl-L-arginine (L-NMMA) and the more abundant asymmetric dimethylarginine (ADMA), which are synthesized and released by the endothelial cells (31). However, chronic exercise did not alter the plasma levels of ADMA in either healthy or heart failure patients (37). Another endogenous NOS inhibitor is PIN, which is expressed inside the diaphragm and selectively inhibits nNOS activity by preventing the dimerization of nNOS monomers (17, 42). We found, however, that mRNA expression of PIN remained unchanged inside the diaphragm during IRL. Although PIN protein levels were not determined, our mRNA results suggest (but do not prove) that PIN does not inhibit diaphragm NOS activity.

ET-1 also inhibits NOS activity (22). Acute exercise increases ET-1 concentration in the plasma (34). Moreover, skeletal muscle fibers endogenously produce ET-1 (16). It is also likely that ET-1 is produced by the endothelial cells of the microvasculature within the muscle, secondary to mechano-transductive stimuli generated during strenuous muscular contractions. Indeed, in human endothelial cells, acute increase in shear stress (30 min) resulted in a significant upregulation of prepro-ET-1 mRNA expression (36). We found that ET-1 as well as ET-3 mRNA expression inside the diaphragm increase significantly during IRL (Fig. 3). Although endothelin protein levels were not determined, we propose (but cannot prove) that enhanced endothelin production inside the diaphragm might have played a role in attenuating NO production. An alternative but not mutually exclusive explanation for the decreased NO derivatives levels in the diaphragm secondary to IRL is augmented NO washout by the increased blood flow to the diaphragm induced by the strenuous diaphragmatic contractions.

Although our results do not directly answer why IRL leads to reduced diaphragmatic NO derivatives levels, one can predict several advantages gained by this response such as: 1) removal of a significant inhibitory influence exerted by NO on diaphragmatic contractility, mediated through cGMP-dependent and cGMP-independent mechanisms involved in excitation-contraction coupling and sarcoplasmic reticulum Ca2+ flux (42) and through the direct inhibition of myosin ATPase activity (39); and 2) endogenous NO synthesis reversibly inhibits mitochondrial enzymes such as cytochrome c oxidase (13). Thus reduction in NO synthesis during IRL is likely to augment mitochondrial respiration and to protect sarcoplasmic reticulum calcium release in response to muscle membrane depolarization.

Regulation of NOS protein expression. We report here for the first time that diaphragmatic NOS protein expression increases significantly during IRL acute resistive loading. These results are not similar to our (15) previous study, which failed to show any significant alterations in diaphragmatic NOS protein expression after 3 h of IRL. We attribute this difference between the two studies to the fact that higher inspiratory loads were actually achieved in the current study compared with our previous study. Indeed, in our (15) previous study, the animals were developing 10 cmH2O average peak tracheal pressure compared with ~35 cmH2O developed in the current study. Prolongation of IRL to 6 h resulted in a further increase in nNOS protein expression. More impressively, iNOS protein expression was also upregulated (400% increase over quietly breathing animals). This is the first report documenting increases in NOS isoform protein expression in striated muscle secondary to acute increases in activity. Previous reports (4, 44, 46) have found increased nNOS and eNOS expression after chronic exercise training (running or swimming) in limb and ventilatory muscles, where enough time for adaptation is allowed, which is not the case for acute exercise. Our current results suggest that nNOS protein expression (the major muscle isoform under resting conditions; Ref. 42) is augmented in the diaphragm secondary to increased activation in a load- and time-dependent manner and that repeated periodic activation (as occurring with chronic exercise training) is not prerequisite for nNOS protein upregulation. By comparison, eNOS expression exhibited similar levels of upregulation after 3 and 6 h of IRL. The reason for the lack of the time-dependence of eNOS upregulation despite the continuous presence of the stimulus (diaphragm activation) is not known. However, it is tempting to speculate that it might be due to the concurrent time-dependent augmentation of TNF-{alpha} expression within the diaphragm of the rats we observed (47), given that TNF-{alpha} decreases eNOS promoter activity as well as eNOS mRNA stability in a time- and dose-dependent manner (49).

The increase in iNOS protein expression in the diaphragm after 6 (but not 3) h of IRL is a novel finding in this study, since even chronic exercise training failed to upregulate iNOS in limb muscles in previous reports (44). The iNOS protein is expressed in skeletal muscles at low levels, and its expression is induced in inflammatory conditions such as sepsis (30) or endotoxemia (8). After LPS administration, iNOS originates from both inflammatory cells infiltrating the perivascular spaces of the diaphragm and resident myocytes. Although our study did not assess the cellular source of iNOS, inflammatory cells can rather safely be excluded, since MPO activity (an index of neutrophil activation and thus indirect evidence for neutrophil influx) did not increase in the diaphragm secondary to IRL. The stimulus for iNOS upregulation during IRL is not known. The fact that iNOS expression in skeletal myocytes is induced significantly in response to a mixture of interferon-{gamma}, IL-1beta, and TNF-{alpha} (48) and that IRL elicits significant upregulation of these cytokines within the diaphragm (47) suggests that iNOS induction in the diaphragm secondary to IRL was the result of upregulation of proinflammatory cytokines. The lack of iNOS induction after 3 h of IRL might be due to insufficient local levels of interferon-{gamma}, IL-1beta, and TNF-{alpha} since the expression of IL-1beta and TNF-{alpha} within the diaphragm was significantly lower at 3 h than after 6 h of IRL (47) and the expression of interferon-{gamma}, a prerequisite cytokine for iNOS induction, was upregulated only after 6 h of IRL (47).

Protein tyrosine nitration in the diaphragm. We identified for the first time several tyrosine-nitrated proteins inside the diaphragm including three members of the glycolysis pathway, enolase, aldolase, and GAPDH. Enolase catalyzes the conversion of 2-phosphoglycerate into phosphoenolpyruvate. Posttranslational modifications of enolase have been linked to several pathologies including the brain of Alzheimer's disease patients and the aging skeletal muscle (24). Aldolase catalyzes the hydrolysis of fructose 1,6-bisphosphate into dihydroxyacetone phosphate and GAPDH. Induction of iNOS in the liver and lung of septic animals is associated with increased aldolase tyrosine nitration (3). Moreover, aldolase is strongly tyrosine-nitrated in limb and cardiac muscles of aged rats (24). GAPDH catalyzes the oxidation of GAPDH to 1,3-bisphosphoglycerate in the second phase of glucose catabolism. Tyrosine nitration of GAPDH has been identified in lung and liver samples of septic rats (3) and in aged skeletal and cardiac muscles (24).

Myosin heavy chain is also tyrosine-nitrated in the diaphragm. This observation, along with those of Kanski et al. (25), which documented tyrosine nitration of myosin light chain, troponin-C, tropomyosin, desmin, and actinin, suggests that endogenously generated peroxynitrite may target muscle contractile proteins and may explain the decline in muscle contractility in response to acute exposure to peroxynitrite (43). Finally, we report here that CAIII is one of the strongly tyrosine-nitrated proteins inside the diaphragm. CAIII is a highly abundant protein in skeletal muscles and functions mainly as a CO2 hydratase, thereby facilitating CO2 diffusion across the sarcolemma.

An interesting observation in this study is that tyrosine nitration of aldolase and enolase declined significantly during IRL and was associated with the reduction in NOx levels. It is worth noticing that the intensity of tyrosine nitration of other proteins remained unchanged during IRL. We have no clear explanation for this observation, but we speculate that the intensity of tyrosine nitration of a given protein is dependent on several factors including the chemical selectivity of the nitrating agent, the turnover rate of tyrosine-nitrated proteins, and the possibility of denitration of tyrosine-nitrated proteins by a putative enzyme called "denitrase" (23). This selective reduction in tyrosine nitration of enolase and aldolase might be interpreted as a protective mechanism to reduce the inhibitory influence of peroxynitrite on their enzymatic activity, thereby enhancing the glycolysis rate and improving ATP production during increased diaphragmatic activity.

In summary, our results indicate that increased diaphragm activation secondary to IRL is associated with significant decline in diaphragmatic NO levels and protein tyrosine nitration of enolase and aldolase. These effects are not mediated by reduction in NOS isoform expression since protein levels of the three NOS isoforms increase in the diaphragm in a time-dependent fashion. This reduction in diaphragm NO levels might represent a compensatory mechanism by which diaphragmatic contractility and metabolism are protected from the negative effects of endogenous NO synthesis.


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 ABSTRACT
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This study is supported by grants from the Canadian Institutes of Health Research and the Canadian Cystic Fibrosis Foundation (S. N. A. Hussain) and an Operational Programme for Education and Initial Vocational Training (EPEAEK) Pythagoras I Grant of the European Union (T. Vassilakopoulos).


   ACKNOWLEDGMENTS
 
We are grateful to L. Franchi for technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: Corresponding author: S. N. A. Hussain, Rm. L3.05, Royal Victoria Hospital, 687 Pine Ave. West, Montreal, Québec, Canada H3A 1A1 (e-mail: sabah.hussain{at}muhc.mcgill.ca)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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