Hum. Reprod. Advance Access originally published online on February 16, 2006
Human Reproduction 2006 21(6):1605-1611; doi:10.1093/humrep/dei500
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Regulation of inducible nitric oxide synthase in post-operative adhesions
1 Department of Obstetrics and Gynecology, The C.S. Mott Center for Human Growth and Development and 2 Department of Biochemistry and Molecular Biology, Wayne State University School of Medicine, Detroit, MI, USA
3 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, The C.S. Mott Center for Growth and Development, Wayne State University School of Medicine, 275 E. Hancock, Detroit, MI 48201, USA. E-mail: gsaed{at}med.wayne.edu
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Abstract |
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BACKGROUND: The deficiency of the inducible nitric oxide synthase (iNOS) substrate, L-arginine (L-Arg), the co-factor tetrahydrobiopterin (H4B) or molecular oxygen may lead to lower NO levels, which enhances the development of adhesion phenotype. METHODS: We utilized high-performance liquid chromatography (HPLC) and immunoprecipitation with nitrotyrosine antibody to determine the levels of H4B, citrulline and protein nitration in fibroblasts established from normal peritoneal and adhesion tissues. RESULTS: The level of H4B was dramatically attenuated in adhesion fibroblasts. The immunoprecipitation with nitrotyrosine antibody revealed higher protein nitration in adhesion compared with normal fibroblasts. There were higher accumulations of citrulline in adhesion fibroblasts as compared with normal fibroblasts. In addition, peritoneal fibroblasts treated with 2% oxygen for 24 h and implanted back into the peritoneal cavity of the rats exhibited marked increase in severity of adhesion as well as extensive distribution involving many sites and organs. CONCLUSIONS: Control of the catalytic activity of iNOS in adhesion fibroblasts may be because of subsaturating amounts of L-Arg and H4B which allow iNOS to generate a combination of reactive oxygen species in addition to NO, thereby influencing NO bioavailability and function.
Key words: adhesions/L-arginine/nitric oxide synthase/surgery/tetrahydrobiopterin
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Introduction |
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Post-operative adhesions occur in the overwhelming majority of patients after laparotomy and laparoscopy. Adhesions are believed to contribute to infertility in 40% of infertile couples and are the most common cause of small bowel obstruction that may occur in either the immediate post-operative period or as late as decades post-surgically.
We have previously begun to characterize differences between human fibroblasts isolated from normal peritoneum and from adhesions and have identified phenotype differences between these two cell types from the same patients (Saed et al., 2001
). Specifically, adhesion fibroblasts have been identified to have a reduction in nitric oxide (NO) levels in addition to other differences that include the following: (i) reduction in both the ratio of tissue plasminogen activator/plasminogen activator inhibitor-1 and in the rate of apoptosis under hypoxic conditions; and (ii) a greater ability to produce the inflammatory cytokine transforming growth factor beta and extracellular matrix molecules than normal peritoneal fibroblasts.
NO is generated by a family of enzymes termed NO synthases that catalyse the formation of citrulline and NO from L-arginine (L-Arg), NADPH and O2 through the formation of N-hydroxy-L-arginine as an intermediate (Stuehr, 1997). In biological systems, released NO may serve as a ligand or substrate, an oxidant or antioxidant or an inhibitor or activator (Stone and Marletta, 1996; Abu-Soud and Hazen, 2000; Abu-Soud et al., 2001). Thus, in a given circumstance, the precise function of NO is driven by its bioavailability, the timing of its release and the bioavailability of its scavengers (Lancaster, 1997). Under normal conditions, NO is typically generated in small amounts and operates as a signalling molecule, but its overproduction or deficiency may cause a series of diseases ranging from asthma to cardiovascular diseases (Moncada, 1994; Persson et al., 1994; Darley-Usmar et al., 1997). Previously, our laboratory and other laboratories have demonstrated that accumulation of NO during steady-state catalysis leads to NO feedback inhibition through the formation of a stable Fe–nitrosyl complex (Galijasevic et al., 2003; Hurshman and Marletta, 1995; Abu-Soud et al., 2000). In the absence of L-Arg and tetrahydrobiopterin (H4B), NO binds to the nitric oxide synthase (NOS) heme iron at a near diffusion rate and generates a five-co-ordinate Fe(II)–NO complex that inhibits the catalytic activity of the enzyme. Under these circumstances, Fe(II) is co-ordinated to four porphyrin nitrogens and one NO molecule, whereas the sixth co-ordinate site opposite NO is free.
To understand how iNOS functions at the site of peritoneal injury and the factors that modulate its activity and functions, we utilized HPLC methods to determine the level of L-Arg and H4B, as well as the by-product citrulline. On the basis of these results, we conclude that lower NO levels in adhesion fibroblasts are because of a combination of effects that include attenuation of levels of both L-Arg and H4B and disturbance in the L-Arg recycling pathway. Understanding the catalytic function of iNOS is important as it will help delineate the mechanism of post-operative adhesion development and thereby reveal new pathways for pharmacological modulation.
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Materials and methods |
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Source and culture of human fibroblasts
As previously described (Saed et al., 2001
), normal parietal peritoneal tissue from the anterior abdominal wall lateral to midline incision and adhesion tissue were excised from patients undergoing laparotomy for pelvic pain, at the initiation of the surgery following entry into the abdominal cavity. Normal peritoneum was at minimum three inches from any adhesions. Subjects did not have an active pelvic or abdominal infection and were not pregnant. All patients gave informed written consent to tissue collection, which was conducted under a protocol approved by the Wayne State University Institutional Review Board. Harvested tissue samples from five women were immediately placed in standard media [Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS), 2% penicillin and streptomycin]. Tissues were cut into small pieces in a sterile culture dish and transferred into another fresh T-25 flask with 3 ml of dispase solution (2.4 U/ml; GIBCO BRL, Invitrogen Corporation, Life Technologies, Carlsbad, California). The flasks were incubated overnight at 37°C in an environ-shaker (LAB-LINE Instruments, Barnstead International, Dubuque, Iowa). The samples were then centrifuged for 5 min at 1400 g, transferred into a fresh T-25 flask with pre-warmed DMEM and placed in a 37°C incubator (95% air and 5% CO2); outgrowth of fibroblasts generally took 2 weeks. Once confluence was reached, the cells were transferred to 90 mm tissue culture dishes and cultured in standard media with 10% FBS. Thereafter, the confluent dishes were sub-cultured by trypsinization (1:3 split ratio). Studies were conducted using passage 3–5 cells to maintain comparability.
l-Arg and citrulline analysis
Analysis of citrulline and L-Arg was performed on a Shimadzu HPLC system, adopting a protocol of Abu-Soud et al. (2000) with modification. Briefly, four volumes of cold methanol were added to cell extract of either adhesion or normal fibroblast pellets. Precipitated protein was removed by centrifugation at 12 000 g. The amino acids in 20 µl of the filtrate were pre-column derived by adding 80 µl of o-phthaldialdehyde (OPA) reagent solution (35 mM OPA, 10 v/v MeOH, 1 v/v mercaptoethanol in 0.1 M sodium borate, pH 10). After 2 min of derivation, a 50 µl sample was injected onto a Prevail C18, 5 µm particle size, 150 ± 4.6 mm reverse-phase HPLC column. The column was eluted at a flow rate of 0.9 ml/min with gradients of buffers A and B (buffer A = H2O and buffer B = methanol) as noted. The solvent gradient was 0–10% B at t = 5–12 min, then 10–95% B at t = 12–20 min. This composition was maintained until t = 28 min before being reduced to the initial 0% B composition. Under these conditions, OPA-derived citrulline and L-Arg were completely resolved and eluted at 13 and 22 min, respectively, and were detected using an RF-10A XL fluorescence spectrophotometer set at 360 nm excitation and 455 nm emission. Citrulline and L-Arg were quantified by a calibration curve prepared on the same day as sample analysis. Each filtrate was analysed in triplicate.
Tetrahydrobiopterin analysis
Measurement of intracellular biopterin derivatives
Fibroblast (adhesion and normal) monolayers were detached with trypsin/EDTA and resuspended in phosphate-buffered saline (PBS) (pH 7.4). Aliquots of 2–5 x 106 cells were centrifuged, and the pellets were mixed with 100 µl of oxidant solution (0.02 M KI/0.5% I2 in 0.1 M HCl or 0.02 M KI/0.5% I2 in 0.1 M NaOH). After sonication on ice, aliquots for the determination of proteins (BCA, Pierce Biotechnology, Inc., Rockford, Illinois) were taken, and the homogenates were incubated for 1 h in the dark at room temperature. Subsequently, 10 µl of HCl (1 M) was added to samples oxidized in base, the precipitates were removed by centrifugation at 12 000 g and excess iodine was destroyed by the addition of 10 µl of ascorbic acid (0.2 M).
Quantification of biopterin in supernatants was performed as described, with minor modification (Heller et al., 2001). Briefly, 20 µl of the oxidized cell extracts was injected onto a Prevail C18, 5 µm particle size, 150 x 4.6 mm reverse-phase column. Biopterin was eluted with a gradient of solvent A (15 mM potassium phosphate buffer, pH 6.4) and solvent B (methanol) at a flow rate of 0.9 ml/min and detected by an RF-10A XL fluorescence at 350 nm excitation and 440 nm emission (Shimadzu Scientific Instruments, Columbia, Maryland). The amount of H4B was calculated from the difference in biopterin concentrations measured after oxidation in acid (total biopterins) and base (7,8-dihydrobiopterin + biopterin). Biopterin levels were expressed in picomoles/micrograms of cell protein.
Western blot for protein nitration
Cell lysate (1 ml) and 0.25 µg of the appropriate control IgG, together with 20 µl of appropriate suspended (25% v/v) agarose conjugate (protein A/G agarose), were incubated at 4°C for 30 min. The beads were pelleted by centrifugation at 1000 g for 30 s at 4°C. Ten micrograms of primary antibody agarose conjugate (i.e. 5 µl volume of packed beads) was added to 100 µg of total cellular protein cell lysate and incubated at 4°C overnight. The pellet was collected by centrifugation at 1000 g for 30 s at 4°C. The pellet was washed two to four times and resuspended in 40 µl of x2 electrophoresis sample buffer. Samples were boiled for 2–3 min before loading 10 µl into 10% polyacrylamide gel for electrophoresis. Proteins were transferred from the gel to a nitrocellulose membrane using an electroblotting apparatus according to the manufacturer’s protocols. Non-specific binding was blocked by incubating the membrane in 5% non-fat milk in tris-buffered saline tween-20 (TBST) for 30–60 min at room temperature.
Membranes were incubated in primary antibody diluted in TBST for 1 h at room temperature and washed three times for 5 min each with PBS. Membranes were incubated for 45 min at room temperature with horse-radish peroxidase (HRP)-conjugated secondary antibody, diluted to 1:500–1:2000 in 2% non-fat milk. Membranes were developed using enhanced chemiluminescence reagent (Amersham Biosciences, General Electric Company, Fairfield, Connecticut). Scanning densometer was used to determine the ratio of intensity of each band relative to 
-actin. Densometric analysis of gel bands was performed using NIH image analysis program.
Rat tissue biopsies for establishment of peritoneal fibroblasts
Sprague–Dawley female rats were subjected to anaesthesia (i.m. ketamine and rompun at the aforementioned dosages). Each animal’s abdomen was shaved, prepped with betadine and then cleansed with 70% isopropanol. Individual sterile drape was placed on the subject, with only its abdomen being exposed. The surgeon was unaware of assigned animal grouping. An approximately 2 cm midline laparotomy immediately beneath the xyphoid process was made. One punch biopsy (2–3 mm) was obtained from the peritoneum in the left upper quadrant at least 1 cm lateral to the midline incision.
The muscle was closed with 3-0 vicryl (Ethicon, Somerville, NJ, USA), and the skin was closed with metal surgical clips. The rats were observed as they recovered and were returned to their respective cages. Rat chow and water was, again, ad lib. Primary cultures of peritoneal fibroblasts were established as previously described (Saed et al., 2001).
Creation of the adhesion phenotype in vitro
Fibroblasts from normal peritoneum were grown in DMEM media. Before transfer, fibroblasts were cultured in either normoxic (20% O2) or hypoxic (2% O2) conditions for 24 h, as previously described (Saed et al., 2001).
Caecal abrasion
On day 0, standardized caecal abrasions were carried out in a fashion that has been previously described and standardized. Anaesthesia again consisted of i.m. ketamine and rompun at the aforementioned dosages. Each animal’s abdomen was shaved, prepped with betadine and then cleansed with 70% isopropanol. Individual sterile drapes were placed on the subject, with only its abdomen being exposed. The surgeon was unaware of assigned animal grouping. An approximately 3 cm midline laparotomy adequately exposed the caecum. The caecum was gently externalized using sterile cotton swabs (Harwood Products, Guilford, ME, USA) and placed onto a raised platform. A sterile polyvinyl chloride (PVC) mat containing a 1.9 cm diameter hole was placed over the exposed caecum.
To create the abrasions, an abrading device that has been used in prior rat caecal abrasion studies, which employs a rotating, motor-driven, constant-force, 70 g metal spline shaft, was used. The shaft had attached at its tip a rubber septum containing a bound, stretched, flat sterile, 8-ply all-cotton gauze surface (Johnson & Johnson, Arlington, TX, USA). The shaft with attached cotton gauze abrading surface was lowered onto, and contacted, the caecum through the 1.9 cm diameter hole in the PVC matting. A researcher blinded to rat grouping assignment was operating the abrading device for all animals. The rats were abraded in random order. Thirty revolutions in two separate caecal locations, the midportion of both the ventral and the dorsal caecal surfaces, were used to produce the standardized lesions. The caecum was returned to the abdomen. The muscle was closed with 3-0 vicryl (Ethicon), and the skin was closed with metal surgical clips. The rats were observed as they recovered and were returned to their respective cages. Rat chow and water was, again, ad lib. Powder-free gloves were used throughout all procedures.
Rat treatments
One million cells in 5 ml of media were placed over the caecum after abrasion in the abrasion group, using a 28G needle. Two groups of six female, 226–250 g, Sprague–Dawley rats (Charles River Laboratories, Wilmington, MA, USA) were evaluated.
Necropsy
Seven days after abrasion, necropsies were performed and adhesions were scored. Immediately after phlebotomy, the abdomen was opened in the midline above the prior incision, the diaphragm was visualized and bilateral pneumothoraxes were created. A blinded observer evaluated the formation of adhesions between the sidewall and the caecum. The presence or absence of adhesions at each site was noted, and the extent of adhesion coverage was scored as follows: 0, clean, no adhesions; 1, adhesions on <50% of the caecum; and 2, adhesions on 50–100% of the caecum. Adhesion severity was scored as 0, clean, no adhesions; 1, filmy adhesions; and 2, dense vascular adhesions.
Statistical analysis
All data were analysed using SPSS for Windows 13.0 (SPSS, Chicago, IL, USA). Comparisons between normal and adhesion fibroblasts were made using paired t-tests for H4B concentration, total biopterin, L-Arg and L-citrulline. As Western blot ratios (nitrotyrosine) are non-parametric data, they were analysed with a Wilcoxon signed rank test. The effects of hypoxia on adhesion formation were analysed using a Fisher’s exact test comparing normoxia versus hypoxia conditions on adhesion scores (positive/negative). Power analysis to determine whether sample size was adequate was conducted for each dependent measure.
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Results |
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To investigate more closely the mechanism of action by which adhesion fibroblasts down-regulate the NOS catalytic activity, we thought first to monitor the concentration of both H4B and the total biopterin in freshly harvested adhesion and normal fibroblast cells. As shown in Figure 1, significant differences in both H4B and total biopterin concentrations exist between the two cell types. Normal peritoneal fibroblasts have significantly higher (mean ± SD) H4B and total biopterin levels (13.9 ± 1.4 and 20.3 ± 1.3 pmol/mg protein) as compared with adhesion fibroblasts (6.7 ± 0.2 and 2.5 ± 0.2 pmol/mg protein), respectively (Figure 1).
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With the use of the same methods, we also determined the content of the NOS substrate, L-Arg, and the product, citrulline, in both adhesion and normal fibroblast cells. Significant differences in the levels of both L-Arg and L-citrulline were identified to exist between the two cell types. Normal peritoneal fibroblasts have significantly higher L-Arg levels (35.3 ± 0.7 nmol/mg protein) as compared with adhesion fibroblasts (29.4 ± 1.7 nmol/mg protein; Figure 2). Additionally, L-citrulline was significantly lower in normal peritoneal fibroblasts (10.4 ± 0.7 nmol/mg protein) as compared with adhesion fibroblasts (28.8 ± 0.9 nmol/mg protein; Figure 2).
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To determine a potential function for iNOS system in primary cultures of fibroblasts established from normal peritoneal and adhesion tissues of the same patient, we used an immunoprecipitation assay, utilizing nitrotyrosine antibody, to determine differences in the levels of nitration between the two cell types. Significant differences in the levels of nitrotyrosine were identified to exist between the two cell types. Normal peritoneal fibroblasts have significantly lower protein nitration (median = 1.975) as compared with adhesion fibroblasts (median = 3.15; Figure 3).
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To determine whether normal peritoneal fibroblasts exposed to hypoxia induced worsened post-operative adhesions, primary cultures of fibroblasts were obtained from the peritoneum of six female Sprague–Dawley rats as described in Materials and methods. Primary cultures of peritoneal fibroblasts were established from each rat as described in Materials and methods. The rats underwent caecal abrasion immediately before the injection of normal peritoneal fibroblasts (1.0 x 106 cells) exposed to normoxia (n = 2) or 2% O2 hypoxia (n = 4) for 24 h in the peritoneal cavity of the rat from which they were established from. Rats receiving fibroblasts exposed to normoxia manifested adhesion development consistent with caecal abrasion alone (primarily filmy and avascular), with the adhesions limited to attachments to the sites of abrasion of the caecum. In dramatic contrast, rats receiving fibroblasts exposed to hypoxia exhibited dramatic increase in severity (thickness, vascularity and ubiquity) of adhesions as well as extensive distribution involving not only the sites of caecal abrasion but also other locations throughout the abdominal cavity including parts of intestine, liver, parietal peritoneum and portions of the reproductive tract (Figure 4).
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Power analysis was conducted for each of the measures and observed to be greater than 0.8 for all measures.
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Discussion |
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Molecular oxygen is an essential substrate for iNOS; its deficiency under a pathophysiological condition such as hypoxia may limit NO production in adhesions. In this report, we have shown that adhesion fibroblasts, induction of the iNOS gene expression, is associated with attenuation in L-Arg and/or H4B levels, which leads to iNOS uncoupling. This process, in turn, may significantly contribute to the lower NO levels observed in adhesion fibroblasts. Reduction in NO levels may significantly contribute to the enhancement in iNOS dimerization (Albakri and Stuehr, 1996
; Chen et al., 2002), shift in the iNOS KmO2 value (Galijasevic et al., 2003; Abu-Soud et al., 2000, 2001) and enhancement in reactive oxygen species levels (Rosen et al., 2002; Werner et al., 2003). Consistent with this concept, rats that received fibroblasts exposed to hypoxia exhibited marked increase in severity of adhesions as well as extensive distribution involving not only the sites of caecal abrasion but also other parts of the intestine, liver, parietal peritoneum and parts of the reproductive tract (Figure 4). In contrast, rats receiving fibroblasts exposed to normoxia manifested adhesion development consistent with caecal abrasion alone, with the adhesion limited to attachments to the caecum. Adhesion severity was evaluated based on the thickness, vascularity and ubiquity (Figure 4). Recently, we have demonstrated that iNOS expression is up-regulated in response to hypoxia in primary cultures of fibroblasts established from normal peritoneal and adhesion tissues. This increase in iNOS expression did not correlate with NO levels and significantly increased programmed cell death (apoptosis) of these cells. Thus, alteration in iNOS level and activity may trigger adhesion development. Collectively, NO concentrations in normal fibroblasts appear to be sufficiently high to prevent adhesion development, whereas NO bioavailability in adhesion fibroblasts is lacking, most likely because of the perturbation in the iNOS cycle, and is insufficient to prevent the development of the adhesion phenotype.
Both L-Arg and H4B play a crucial role in NOS coupling, and their deficiency allows the enzyme to generate the free radical 
, instead of NO (Galijasevic et al., 2003; Rosen et al., 2002; Werner et al., 2003). The results of this report extend our recent findings across phylogenetic boundaries and demonstrate, for the first time, that the lack of NO synthesis in adhesion fibroblasts is because of a decrease in intracellular L-Arg and H4B. This decrease in both components in adhesion fibroblasts allows the attenuation of iNOS coupling and leads to the production of peroxynitrite (ONOO–), which may directly enhance protein tyrosine nitration. This is consistent with our finding that adhesion fibroblasts manifested increased tyrosine nitration (Figure 3). In addition to these abnormalities, our current results also show that the adhesion fibroblasts have considerably elevated citrulline accumulation compared with normal. These findings may suggest a partial disturbance in the transcellular metabolic pathway to recycle L-citrulline back to L-Arg, a process that may also contribute to the inefficiency of NOS in generating NO. Additionally, citrulline accumulation is a result of several pathways other than just the iNOS system. One could look at the regulation of enzymes involved in citrulline metabolism, such as the urea cycle enzymes.
A kinetic model that incorporates our present and previous published results is presented in Figure 5. The heme prosthetic group of NOS performs a crucial role in ligand binding as well as the catalytic function of the enzyme. Its essential function is to promote electron transfer within the catalytic domain of the enzyme during catalysis. In normal fibroblasts, the presence of saturated amounts of both L-Arg and H4B increases the NOS coupling, and the bioavailability of NADPH and O2 leads to NO synthesis and citrulline production. Upon binding, NADPH donates electrons to the heme through flavin mononucleotide (FMN) and flavin adeninedinucleotide (FAD) through a two-step mechanism that involves the formation of N
–hydroxy-L-Arg as an intermediate, generating NO and citrulline as end products. In contrast, in adhesion fibroblasts, subsaturating amounts of either H4B or L-Arg decrease NOS coupling and result in the generation of a mixture of reactive oxygen species (e.g. , H2O2 and ONOO–) in addition to NO (Figure 5). ONOO– and its conjugate acid, peroxynitrous acid (ONOOH), are capable of promoting both protein nitration and initiation of lipid peroxidation, processes known to occur during tissue injury associated with inflammation in vivo.
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Here, we demonstrated that tyrosine nitrated proteins are present in both adhesion and normal fibroblasts. Comparisons show that patterns of protein nitration are different, with more nitrated proteins in adhesion fibroblasts. These cytotoxic species may contribute significantly to the development of the adhesion phenotype in the fibroblasts, which populates the injured peritoneum during the peritoneal healing process. Indeed, there is ample evidence that H4B deficiency provokes NOS-derived /H2O2 production in conditions of oxidative stress (Werner et al., 2003).
Through in vitro studies, roles of H4B and L-Arg in re-shaping the architecture of the NOS heme pocket geometry, narrowing the heme pocket, stabilizing ligand binding to the heme prosthetic group and strengthening the iron–thiolate bond have been revealed (Hurshman and Marletta, 1995; Stuehr, 1997; Abu-Soud et al., 1998, 2000, 2001). Lines of evidence obtained by a variety of spectroscopic techniques, diatomic ligand binding, as well as site-directed mutagenesis, provided clear evidence that L-Arg and H4B, upon binding to NOS, have remarkable impact on the observed spectral properties and functional characteristics (Chen et al., 2002). Studies with NOS isoforms demonstrated that NO binds L-Arg- and H4B-free NOS-Fe(II) or to recombinant NOS mutants that display high Kd values and low affinity of H4B at a near diffusion control rates, thereby forming an unstable six-co-ordinate complex, which is immediately converted to its respective five-co-ordinate complex (Figure 5) (Abu-Soud et al., 1998; Migita et al., 1997; Huang et al., 1999). Providing saturated amounts of L-Arg and H4B could prevent the Fe–S bond breakage and could, subsequently, restore the catalytic activity of NOS (Figure 5) (Abu-Soud et al., 1998; Migita et al., 1997; Huang et al., 1999), indicating an important role of these molecules in stabilizing the thiol–iron bond in the enzyme. A recent study with iNOS using rapid-quench electron paramagnetic resonance (EPR) techniques illustrated that bound H4B is oxidized to a radical form when ferrous iNOS reacts with O2, providing the first direct evidence that H4B displayed a role in electron transfer in the NOS reaction (Hurshman et al., 1999; Wei et al., 2001). Earlier studies by Abu-Soud et al. (1997) are consistent with these observations, which have suggested that H4B significantly accelerates the decay of Fe(II)–O2 even in the presence of L-Arg (Abu-Soud et al., 1997, 2000, 2001). Thus, H4B and L-Arg play a crucial role in creating the desired conformational environment of NOS heme iron that is required for catalysis (Hurshman and Marletta, 1995; Abu-Soud et al., 1997, 2000; Chen et al., 2002).
Citrulline is a non-essential amino acid and is a precursor for the synthesis of L-Arg. Its conversion to L-Arg is a complex process that involves aspartic acid, as well as two essential enzymes, ornithine transcarbamoylase and argininosuccinate synthetase (Wu and Morris, 1998). Despite its role in L-Arg synthesis, citrulline is also an essential component of the urea cycle, promotes energy and fuels the immune system (Wu and Morris, 1998). Thus, in adhesion fibroblasts, an increased and profound disturbance in catabolism of L-Arg may lead to citrulline accumulation and, subsequently, a decrease in NO synthesis. These observations suggest the tempting speculation that supplementation with citrulline and L-Arg may play a beneficiary role in reduction of post-operative adhesion development.
Long-term changes in NO production by hypoxia have been previously reported (Liao et al., 1995; Melillo et al., 1995). The massive appearance of post-operative adhesions in our well-developed caecal abrasion rat model utilizing rats treated with hypoxic cells noted in this study indicated either post-translational modification of the iNOS gene or a change in NO consumption by scavengers. It is unlikely that rates of NO consumption by superoxide would change to varying inspired oxygen levels. Rather, change in NO production is likely, based on our current results, that hypoxia directly affects iNOS activity in our cell culture system. Hypoxia causes potential alteration in cellular respiration and metabolism that may affect cellular levels of co-factors required for NOS activity. Hypoxia not only reduces H4B and L-Arg levels but also reduces NADPH levels, with significant decrease in NADPH at oxygen levels <15 µM (Masters et al., 1983; Tribble and Jones, 1990; Wakita et al., 1995). In addition, our previous in vitro studies utilizing purified iNOS indicated that the enzyme catalytic activity depends on O2 concentration despite excess concentration of L-Arg and H4B. However, in the absence of NO synthesis, the KmO2 values dramatically decreased (Abu-Soud et al., 2000, 2001).
NO is freely diffusable and displays a remarkable capacity in activating or inhibiting many hemoproteins and non-hemoproteins (Stone and Marletta, 1996; Lancaster, 1997; Cooper, 1999; Abu-Soud and Hazen, 2000; Abu-Soud et al., 2001). NO and O2 compete on the iNOS heme iron. In related studies, we have previously shown that in the presence of NO synthesis, the KmO2 value significantly enhanced, suggesting an important role of O2 in directly regulating iNOS activity in vivo (Abu-Soud et al., 2000, 2001). A steady level of NO production is maintained under normoxia and hypoxia, although NO production was decreased under hypoxia. The oxygen concentration in intact tissues ranges from 1 to 150 µM (Vanderkooi et al., 1991). An increase in iNOS gene expression and NO level is associated with many inflammatory diseases, such as atherosclerosis and asthma (Moncada, 1994; Persson et al., 1994; Darley-Usmar et al., 1997), but in adhesion fibroblasts, is associated with a significant decrease in NO levels.
In summary, the reduced NO levels in adhesion fibroblasts are associated with reduced H4B and L-Arg levels, which alter NOS efficiency enhancing generation. It is tempting to speculate that delivery of supplementary H4B and L-Arg to patients before surgery and during their recovery may be a novel consideration as a therapeutic option for reducing post-operative adhesion development and their clinical sequelae.
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Acknowledgements |
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This work was supported by NIH grant number HL066367 to H.M.A.-S.
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References |
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Submitted on 30 June, 2005; resubmitted on 29 September, 2005, 22 November, 2005, 6 December, 2005 ; accepted on 22 December, 2005
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