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Abstract |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Nitric oxide synthases (NOS) convert L-arginine to nitric oxide in the presence of tetrahydrobiopterin (BH4). Two constitutive isoforms of NOS exist in normal skeletal muscle fibers, however, the existence of a third, the inducible isoform (iNOS), has never been detected in these fibers in vivo. Therefore, we assessed the influence of in vivo endotoxemia on skeletal muscle expression of constitutive and inducible NOS isoforms and GTP cyclohydrolase I, the rate limiting enzyme of BH4 synthesis. Two groups of rats were infused i.p. either with E. coli endotoxin (20 mg/kg, LPS group) or saline (saline group). Animals were killed 6 h later and the ventilatory and limb muscles were quickly frozen. Endotoxin infusion elicited a significant rise in NOS activity of the diaphragm, intercostal, soleus and gastrocnemius muscles. Reverse transcription-polymerase chain reaction (RT-PCR) on muscle total RNA detected very low expression of iNOS and GTP cyclohydrolase I mRNA in the saline group, but significant upregulation of both enzymes was found in the ventilatory and limb muscles of the LPS group. Immunoblotting detected no iNOS protein in the saline group but a significant iNOS protein expression was found in the diaphragm, intercostal and soleus muscles and to a lesser extent, in the gastrocnemius of the LPS group. Endotoxemia was also associated with increased protein expression of constitutive NOS isoforms mainly in the diaphragm and to lesser extent in the intercostal, gastrocnemius and soleus muscles. We conclude that in vivo exposure to endotoxin leads to differential induction of both iNOS and GTP cyclohydrolase I in the ventilatory and limb muscles.
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Introduction |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Hussain and colleagues (1) and others (2) previously reported that the contractile function of the ventilatory muscles is significantly depressed in endotoxemia or sepsis. Several mechanisms have been implicated in the depressed muscle contractility among which is mismatching of blood flow to metabolic demands as a result of local vascular dysfunction (3, 4). In our more recent study, Hussain (5) described that endotoxemia-induced diaphragmatic vascular dysfunction is due in part to enhanced nitric oxide (NO) release, however, the source of NO release was not identified in that study.
Nitric oxide is a multifunctional molecule which is synthesized from L-arginine by a group of flavo proteins known as nitric oxide synthases (NOS)(6). Three main NOS isoforms have been identified, two of which are constitutively expressed and were initially identified in the endothelial cells (ecNOS, type III) and brain cells (bNOS, type I). The third is an inducible isoform (iNOS, type II) (6). While the requirements for Ca2+ and calmodulin differ between ecNOS and bNOS on one hand and iNOS on the other, the activity of the three isoforms are critically dependent on the presence of L-arginine, NADPH and tetrahydrobiopterin (BH4) (6).
It has recently been established that ecNOS and bNOS are constitutively expressed in skeletal muscle fibers (11, 12). While ecNOS is localized in the mitochondria (13, 14), distinct staining of bNOS was identified at the sarcolemma of mainly type II muscle fibers (12). The exact roles of these two isoforms in regulating muscle function remain unknown, however, augmentation of activity and/or gene expression of these isoforms could explain enhanced NO release in the diaphragm of endotoxemic animals. The most likely source of enhanced NO release in endotoxemia, however, is inducible NOS. Unlike ecNOS and bNOS, the expression of iNOS has never been demonstrated in normal skeletal muscle fibers. Numerous investigators, on the other hand, have found iNOS expression in macrophages, vascular smooth muscle cells, hepatocytes, endothelial and epithelial cells in response to in vivo or in vitro exposure to bacterial endotoxin and/or inflammatory cytokines such as tumor necrosis factor, interleukin-1, or gamma interferon (15). Induction of iNOS in these cells is also known to be associated with induction of GTP cyclohydrolase I, the rate limiting enzyme in the synthesis of BH4 (18). Whether or not these inflammatory cytokines and/or bacterial endotoxin lead to the in vivo induction of iNOS in skeletal muscles remains unknown. To our knowledge, the only published report on the in vivo effects of endotoxin on skeletal muscle NOS activity are those of Szabo and associates (19) and Salter and colleagues (20), in which no change or a slight increase in Ca2+/calmodulin-independent NOS activity of skeletal muscles has been reported.
The main aim of this study was to investigate the acute influence of bacterial endotoxin infusion on total NOS activity and protein expression of various NOS isoforms in the ventilatory and limb muscles. We also assessed the effect of bacterial endotoxin on the expression of GTP cyclohydrolase I in various skeletal muscles.
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Materials and Methods |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Reagents
Materials for L-citrulline assay were obtained from Sigma
(St. Louis, MO). L-[2,33H]-arginine was obtained from Dupont Inc. Western blotting apparatus and gels were obtained
from Novex Inc. (San Diego, CA). Monoclonal anti-iNOS,
anti-ecNOS and anti-bNOS antibodies were obtained from Transduction Laboratories (Lexington, KY). ECL detection kit was obtained from Amersham Corp. 
X174RF/
Hae III DNA marker was obtained from Life Technologies Inc. (Gaithersburg, MD).
Animal Preparation
The procedures were approved by the Animal Research Committee of McGill University. Pathogen-free male Sprague Dawley rats (250-300 g) were studied 1 wk after arrival. Animals were injected i.p. with E. coli lipopolysaccharides (serotype 055:B5, 20 mg/kg LPS group) or normal saline (saline group) and were killed by cervical dislocation 6 h after the injection. Ventilatory and limb muscles were dissected from the animals and quickly frozen in liquid nitrogen. The collected tissues included diaphragm, intercostal, transverse abdominous, gastrocnemius (red and white portions), soleus and tibialis anterior.
L-Citrulline Assay
Details of the assay have been published previously (11). Frozen tissues were homogenized in 6 volumes (w/v) of homogenization buffer (pH 7.4, 10 mM HEPES buffer, 0.1 mM EDTA, 1 mM dithioreitol, 1 mg/ml PMSF, 0.32 mM sucrose, 10 µg/ml leuopeptin, 10 µg/ml aprotinin, 10 µg/ml pepstatin A). The crude homogenates were centrifuged at 4°C for 15 min at 10,000 rpm. The supernatant (50 µl) was added to 10 ml prewarmed (37°C) tubes containing 100 µl of reaction buffer of the following composition: 50 mM KH2PO4, 60 mM valine, 1.5 mM NADPH, 10 mM FAD, 1.2 mM MgCl2, 2 mM CaCl2, 1 mg/ml BSA, 1 µg/ml calmodulin, 10 µM BH4 and 25 µl of 120 µM stock L-[2,33H]- arginine (150-200 cpm/pM). The samples were incubated for 30 min at 37°C and the reaction was terminated by the addition of cold (4°C) stop buffer (pH 5.5, 100 mM HEPES, 12 mM EDTA). To obtain free L-[3H]-citrulline for the determination of enzyme activity, 2 ml of Dowex 50w resin (8% cross linked, Na+ form) were added to eliminate excess L-[2,33H]-arginine. The supernatant was assayed for L-[3H]-citrulline by using liquid scintillation counting. Enzyme activity was expressed in pmol of L-citrulline produced/min/mg total protein. Protein was measured by the Bradford technique with bovine serum albumin (BSA) as standard (Bio-Rad Inc., Hercules, CA). NOS activity was also measured in the presence of 1.5 mM of each EGTA and EDTA which replaced CaCl2 and calmodulin in the reaction buffer and in the presence of 1 mM of NG-nitro-L-arginine methyl ester (NOS inhibitor). Ca2+/calmodulin-dependent NOS activity was calculated as the difference between that measured in the presence of CaCl2 and that measured in EDTA/EGTA buffer. Ca2+/calmodulin-independent NOS activity was calculated as the difference between samples assayed in the presence of EGTA/EDTA and those measured in the presence of NG-nitro-L-arginine methyl ester.
Immunoblotting
Crude homogenate proteins (80 µg) were heated for 15 min at 90°C and then loaded on gradient (4-12%) Tris-Glycine SDS-PAGE. Proteins were electrophoretically transferred onto PVDF membranes and were blocked overnight (4°C) with 5% non-fat dry milk and subsequently incubated with either primary monoclonal anti-iNOS (1:500), monoclonal anti-ecNOS (1:1,000) or monoclonal anti-bNOS (1:1,000) antibodies. This antibody was raised against 21, 20.4, and 22.3 kDa protein fragments corresponding to mouse macrophage iNOS, human endothelial ecNOS and human bNOS sequences. Our extensive preliminary experiments revealed that these antibodies are selective to their corresponding NOS isoforms in rat tissues. Lysate of cytokine-activated murine macrophages, human endothelial cells and human pituitary were used as positive controls. Specific proteins were detected using HRP-conjugated anti-mouse secondary antibody and enhanced chemiluminescence reagents provided with an ECL kit (Amersham Canada, Oakville, ON). The blots were scanned with an imaging densitometer (12-bit precision and 42-µm resolution, Model GS700, Bio-Rad Inc.) and optical densities of protein bands were quantified with a software (SigmaGel, Jandel Scientific, San Rafael, CA). Predetermined MW standards (Novex Inc., San Diego, CA) were used as markers. To assess the site of iNOS expression, we centrifuged crude muscle homogenates at 100,000 × g for 60 min (4°C). The soluble fraction was removed and the particulate fraction was resuspended in homogenization buffer (see above). Fractions were analyzed with immunoblotting along with the crude homogenate sample.
Immunohistochemistry
Muscle tissues were flash frozen in cold isopentane (20 s)
and then immersed in liquid nitrogen and stored at 
80°C.
Air dried cryostat sections (10 µm) were rehydrated with
phosphate-buffered saline (PBS) (pH 7.4, 3-5 min) and
then blocked for 1 h with normal donkey or horse serum
followed by washing with PBS. For accurate detection of
iNOS we used monoclonal (20 µg/ml in PBS containing 1% sovine serum albumin [BSA], Transduction Laboratories) or polyclonal (dilution 1 in 500; Upstate Biotechnology Inc., Lake Placid, NY) antibodies. Sections were
incubated with these primary antibodies for 1 h at room
temperature. After 3 washes in PBS, sections were incubated with biotinylated goat anti-rabbit or horse anti-mouse secondary antibodies followed by treatment with
the avidin-biotin-peroxidase complex (Vector Laboratories,
Burlingame, CA). Sites of immunoreaction were visualized by immersing sections in a solution of diaminobenzidine and hydrogen peroxide. Counerstaining was performed
with hematoxylin (Sigma, Inc.). A similar protocol was
used for negative control sections except that anti-iNOS
antibody was replaced with mouse or rabbit IgG.
Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
Total RNA was extracted from tissue samples following the method described by Chomczynski and Sacchi (21). One µg of total RNA was reverse transcribed using random hexamers and MMLV reverse transcriptase (Life Technologies, Gaithersburg, MD). RT-generated cDNA encoding iNOS, GTP cyclohydrolase I and glyceraldehydes-3-phosphate dehydrogenase (GAPDH, both as an internal standard and positive control) were amplified using PCR. RNA with no clear GAPDH band in the RT-PCR products (30 cycles) was discarded from further studies. Oligonucleotide primers (synthesized in McGill University DNA synthesis facility) for iNOS, GTP cyclohydrolase I and GAPDH are listed in Table 1. These primers were chosen on the basis of the sequence of mouse macrophage iNOS (accession #M84373)(22), rat GTP cyclohydrolase I (23) and rat GAPDH (24). Experimental conditions for all PCR reactions were: initial denaturation at 95°C for 5 min followed by 30 cycles (94°C/1 min, 60°C/1 min and 72°C/1.5 min). This was followed by a final 10 min, 72°C extension. Ethidium bromide-stained 2% agarose gels were used to separate PCR products. PCR products were visualized under ultraviolet light and the optical density of DNA bands were scanned with a densitometer and quantified using SigmaGel software (Jandel Scientific). To verify the accuracy of the amplified sequence, PCR products were cloned in PCRII plasmid (Invitrogen, San Diego, CA) and sequenced in the McGill University DNA sequencing facility.
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Data Analysis
Results of NOS activity are presented as means ± SEM. Differences in NOS between saline and LPS groups for a given muscle and between muscles were compared using two-analysis of variance for repeated measures. Any differences detected were evaluated post hoc using the Neuman-Keuls procedure. P < 0.05 was considered significant. Similar analysis was used to compare optical density of RT-PCR products stained with ethidium bromide.
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Results |
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Abstract
Introduction
Materials & Methods
Results
Discussion
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Figure 1 illustrates the average values of total and Ca2+/ calmodulin-independent NOS activity of crude homogenates of various muscles in both groups. Total NOS varied significantly among different muscles in the saline group with the gastrocnemius showing the highest activity and the soleus having the lowest activity. In all muscles of the saline group, Ca2+/calmodulin-independent NOS activity was negligible. Compared with the saline group, total NOS activity in the LPS group increased significantly in the diaphragm (P < 0.01), intercostal, transverse abdominous, gastrocnemius and soleus muscles (P < 0.05). Tibialis NOS activity in the LPS group, by comparison, was not different from that of the saline group. The rise in total NOS activity in the diaphragm and intercostal muscles was due to both an increase in Ca2+/calmodulin-dependent and independent NOS activities (P < 0.05 compared with saline), whereas only Ca+/calmodulin-dependent NOS activity rose significantly in transverses abdominous, gastrocnemius and soleus muscles (Figure 1).
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Figure 2 illustrates the results of RT-PCR obtained with iNOS (top), GTP cyclohydrolase I (middle) and GAPDH (bottom) primers in different muscles of the saline (left panels) and LPS (right panels) groups. Relatively low expression of iNOS and GTP cyclohydrolase I mRNA was found in the muscles of the saline group. LPS injection resulted in a significant rise in the expressions of both iNOS and GTP cyclohydrolase I mRNA in all muscle samples compared with the saline group (P < 0.01). No difference in GAPDH between the saline and LPS groups was noticed (Figure 2). Protein expression of iNOS in different muscles is shown in Figure 3. In the LPS group, abundant iNOS protein was found in the diaphragm, intercostal and soleus muscles, but weaker expression was evident in the gastrocnemius muscle. No detectable iNOS protein expression was found in the tibialis in the LPS group. In addition, none of the muscles of the saline group showed evidence of iNOS protein (Figure 3). Figure 4 illustrates an immunoblot of crude homogenate, soluble and particulate fractions of the diaphragm of a septic rat. iNOS protein was detected in the crude homogenate as well as in the particulate fraction, whereas no iNOS protein was detected in the soluble fraction. Probing of a similar blot (not shown) with an antibody against a cytosolic protein (Cu/Zn-Superoxide Dismutase; Chemicon International Inc., Temecula, CA) verified the purity of the soluble and particulate fractions.
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Figure 5 illustrates staining with anti-iNOS antibodies of several muscles obtained from control and LPS groups. Extensive staining of numerous muscle fibers were evident in the intercostal (right upper panel) and diaphragm (left upper panel) of the LPS group. Similar staining was also seen in the gastrocnemius and soleus muscles (not shown). In addition to muscle fibers, iNOS staining was observed in white cells such as monocytes infiltrating muscle fibers of the LPS group (left middle panel). Tibialis muscles of the LPS group showed very weak iNOS staining (right middle panel). No staining with iNOS antibodies were observed in the control group (intercostal, right lower panel). Negative control for intercostal muscle obtained from the LPS group is shown in the left lower panel.
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Figure 6 shows changes in ecNOS protein expression in the diaphragm, gastrocnemius and soleus muscles in the two animal groups. Anti-ecNOS antibody detected a single band (~ 130 kDa) in the control muscles. A similar size band was seen in the muscles of the LPS group, however, optical densities of the diaphragm, gastrocnemius and soleus ecNOS bands of the LPS group were 123, 167, and 154% of comparable muscles of the control group, respectively. A similar rise (142% of control) in the intercostal muscle ecNOS protein expression was observed (data not shown).
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The changes in bNOS protein expression of various muscles in the two groups of animals are shown in Figure 7. Anti-bNOS antibody detected in the control group a protein band of ~ 160 kDa which was more prominent in the intercostal and gastrocnemius muscle and less prominent in the diaphragm. Very weak bNOS band was detected in the control soleus muscle (Figure 7). Significant induction of bNOS protein was evident in the diaphragm of the LPS group (optical density of 674% of the control muscle). Less prominent induction of bNOS protein was seen in the intercostal (166% of control) and the gastrocnemius muscles (118% of control). Although very weak bNOS expression was present in the soleus muscle when compared with other muscles, LPS infusion increased bNOS band optical density to 160% of control.
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Discussion |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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The main findings of this study are: (1) Endotoxin infusion elicited a significant increase in skeletal muscle NOS activity. In the diaphragm and intercostal muscles, this increase was due to augmentation of both Ca2+/calmodulin-dependent and Ca2+/calmodulin-independent NOS activity, whereas only Ca2+/calmodulin-dependent nitric oxide synthase activity rose significantly in transverse abdominous, gastrocnemius and soleus muscles. (2) Endotoxin infusion evoked the induction of iNOS and GTP cyclohydrolase I mRNA in the ventilatory and limb muscles. (3) No iNOS protein was evident in normal ventilatory and skeletal muscles but a significant induction of iNOS protein was found in the diaphragm, intercostal and soleus muscles, and to a lesser extent in gastrocnemius muscles. iNOS protein appears to be localized in the particulate rather than the soluble fraction of skeletal muscles samples. (4) LPS infusion was associated with elevation of ecNOS and bNOS expression in various muscles.
Expression of NOS in Skeletal Muscles
The effects of LPS or inflammatory cytokines on muscle NOS activity and protein expression have not been thoroughly investigated. To our knowledge, the only published data in this respect are those of Szabo and associates (19) and Salter and colleagues (20) who reported a small increase in diaphragmatic and no change in skeletal muscle NOS activity 3 and 6 h after endotoxin injection in rats. Our study represents the first comprehensive attempt at correlating the changes in NOS activity, iNOS protein and mRNA expression in various skeletal muscles of endotoxemic rats.
We found that both Ca2+/calmodulin-dependent and independent NOS activities increased significantly, mainly in the diaphragm and to lesser extent in other muscles. While the rise in Ca2+/calmodulin-independent NOS activity could be explained by the induction of iNOS, increasing Ca2+/calmodulin-dependent muscle NOS activity could be attributed to the following factors. First, increased ecNOS and bNOS protein expression could explain the rise in Ca2+/calmodulin-dependent muscle NOS activity. This is particularly true in the diaphragm where bNOS expression was increased by several fold in response to endotoxin infusion (Figure 7). Although we are the first to report that muscle ecNOS and bNOS protein expressions are regulated in endotoxemic animals, we didn't determine whether this regulation is mediated by enhanced transcription rate and/or improved mRNA stability. Nevertheless, our findings of enhanced ecNOS expression in the LPS group confirms previous studies indicating that inflammatory cytokines and LPS enhance NO production by ecNOS in murine brain microvascular, renal endothelial, bovine, porcine aortic and human umbilical vein endothelial cells (25). Unlike ecNOS expression, little is known about the influence of bacterial endotoxin on the expression of bNOS. Lee and colleagues (28) reported that infusion of a relatively small dose of LPS in rats elicited a significant rise in bNOS mRNA in the hypothalamus. Our results suggest that muscle bNOS expression changes in a similar fashion to that of the hypothalamus in response to endotoxin infusion. Second, augmentation of local availability of BH4 could lead to enhanced dimerization of NOS subunits and, hence, promotes NO production by the three NOS isoforms (29). BH4 is synthesized from GTP through several steps requiring GTP cyclohydrolase I (the rate limiting enzyme), 6-pyruvoyl tetrahydrobiopterin synthase and septiapterin reductase. It has been established recently that inflammatory cytokines and LPS in endothelial cells, tumor cells, fibroblasts and hepatocytes induce iNOS and GTP cyclohydrolase I and lead to increased BH4 concentration (18, 30). Although we did not measure BH4 concentration in muscle samples, we propose that significant upregulation of GTP cyclohydrolase I expression is likely to increase BH4 concentration which, in turn, enhances NOS catalytic activity. Third, increased Ca2+/calmodulin-dependent NOS activity in muscles may be due to the induction of a special isoform of iNOS whose activity is dependent on Ca2+ and calmodulin (16). This iNOS isoform was isolated from liver, lung, spleen and colon after the injection of Propionbacterium acnes and E. coli LPS (31). The possibility of induction of this iNOS isoform in our study could not be ruled out because this isoform is indistinguishable in terms of gene and protein sequence from Ca2+/calmodulin-independent iNOS.
Although the induction of iNOS mRNA and protein in vascular cells has received considerable attention, little is known about iNOS induction in cardiac and skeletal muscles. Balligrand and colleagues (32) recently found abundant iNOS mRNA and protein expression in primary culture of adult rat ventricular myocytes after 12 h of exposure to inflammatory cytokines. To our knowledge, there is only one report documenting in vitro iNOS expression in response to inflammatory cytokines in C2C12 cultured skeletal myocytes and myoblasts (33).
We are the first to report the in vivo induction of iNOS in various ventilatory and limb muscles in endotoxemic animals. Our result indicates that iNOS induction which was more strong in the ventilatory muscles compared with limb muscle was associated with induction of GTP cyclohydrolase I. We should point out that while gastrocnemius Ca2+/calmodulin-independent NOS activity was not detectable (Figure 1) and iNOS protein expression in that muscle was weak (Figure 3), significant iNOS mRNA expression was detected with RT-PCR after LPS injection (Figure 2). Similarly high iNOS mRNA expression was detected in the soleus. whereas a small rise in Ca2+/calmodulin-independent NOS activity was detected in that muscle after LPS injection (Figure 1). We attribute the relatively high iNOS mRNA expression in these muscles to the fact that PCR is a very sensitive technique which could detect even a single copy of a given mRNA transcript. In addition, we used 1 µg of total RNA and a total of 30 cycles to amplify iNOS mRNA expression (see METHODS). At these parameters, the concentration of PCR products is not linearly related to the concentration of the template (iNOS mRNA concentration). Thus, the intensity of iNOS PCR products in Figure 2 should be interpreted in qualitative (iNOS induction) and not in quantitative terms.
The molecular mechanisms responsible for the induction of iNOS in skeletal muscles were not assessed in our
study; however, we speculate based on the results obtained from macrophage and endothelial cell studies, that
LPS directly and/or in concert with inflammatory cytokines activates iNOS promoter through complex pathways
involving tyrosine kinases, protein kinase C and the transcription factor NF-
B (34).
Physiological Implications
The functional consequence of enhanced NO production in the function of ventilatory and limb muscles has not been assessed. Recent studies on in vitro isolated muscles indicate that NO exerts an inhibitory effect on muscle contractility (12). On the basis of these results, we argue that iNOS induction and the rise in NO production in endotoxemic animals is likely to have a deleterious effect on ventilatory muscle function and could explain in part the previously described ventilatory muscle failure in endotoxemic and septic animals (1, 2). We propose the following mechanisms through which iNOS expression and increased NO production may depress muscle contractile function. First, NO attenuates mitochondrial respiration by inhibiting several enzymes such as cytochrome oxidase and aconitase (13, 35). Second, NO interacts with superoxide anions leading to the formation of peroxynitrite, a potent radical species which targets various molecules such as thiols, lipids and protein containing aromatic amino acids (36). Several investigators described the formation of peroxynitrite in aorta and lungs of endotoxemic rats. It is possible that iNOS induction in the muscles of endotoxemic rats leads to the formation of peroxynitrite which, in turn, causes depressed muscle contractile function. We recently confirmed the formation of peroxynitrite in the diaphragm of endotoxemic rats by measuring the concentration of nitrotyrosine in diaphragmatic tissue extract (unpublished observation). Third, induction of iNOS in vascular smooth muscle cells could cause depressed vascular reactivity and poor matching of blood flow to metabolic demands in endotoxemic animals. Enhanced NO release has indeed been implicated by Hussain in endotoxin-induced diaphraglmatic dysfunction (5).
In summary, our results indicate that NOS activity increases significantly in the ventilatory and limb muscles of endotoxemic rats. This rise in NOS activity is attributed in part to the induction of iNOS mRNA and protein as well to the induction of ecNOS and bNOS isoforms and GTP cyclohydrolase I, the rate limiting enzyme for synthesizing BH4.
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Footnotes |
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Address correspondence to: Dr. Sabah N. A. Hussain, Room L3.05, Royal Victoria Hospital, 687 Pine Av. West, Montreal, PQ, H3A 1A1 Canada. E-mail: SHUSSAIN{at}RVHMED.LAN.MCGILL.CA
(Received in original form April 9, 1996 and in revised form November 20, 1996).
Acknowledgments: This study is supported by the MRC of Canada and the Heart and Stroke Foundation of Canada. Dr. Hussain is a scholar of the FRSQ (Quebec). The authors are grateful to Ms. J. Long and Ms. R. Carin for their assistance.
Abbreviations
BH4, tetrahydrobiopterin;
BSA, bovine serum albumin;
ECL, enhanced chemiluminescence;
EDTA, ethylenediaminetetraacetic acid;
EGTA, ethylene glycol-bis (
-aminoethyl ether) N,N,N',N'-tetraacetic
acid;
FAD, flavine adenine dinucleotide;
HRP, horseradish peroxidase;
LPS, lipopolysaccharides;
MMLV, moloney murine leukemia virus;
MW, molecular weight;
NADPH, 
-nicotinamide adenine dinucleotide phosphate
reduced form;
NOS, nitric oxide synthase;
PBS, phosphate-buffered
saline;
PMSF, phenylmethylsulfonyl fluoride;
PVDF, polyvinylidene difluoride;
RT-PCR, reverse transcription-polymerase chain reaction;
SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
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References |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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1.
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