Production of IL-1{beta}, hydrogen peroxide, and nitric oxide by colonic mucosa decreases sigmoid smooth muscle contractility in ulcerative colitis

Weibiao Cao,1,2 Claudio Fiocchi,3 and Victor E. Pricolo2

1Department of Medicine and 2Department of Surgery, Rhode Island Hospital and Brown Medical School, Providence, Rhode Island; 3Division of Gastroenterology, Case Western Reserve University School of Medicine, Cleveland, Ohio

Submitted 18 February 2005 ; accepted in final form 15 July 2005


    ABSTRACT
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We have previously shown that sigmoid circular muscle cells from patients with ulcerative colitis (UC) exhibit reduced contraction and Ca2+ signaling in response to the neurotransmitter neurokinin A (NKA) and that IL-1{beta} and H2O2 may contribute to these reduced responses in UC. In addition, we have found that nitric oxide (NO) levels were significantly increased in UC circular muscle. To establish the site of origin for IL-1{beta}, H2O2, and NO, we assembled an in vitro system in which normal or UC mucosa were sealed between two chambers filled with oxygenated Krebs solution. Because the mucosa consists of full-thickness mucosa and submucosa, it is expected that whatever is released into the undernatant from the submucosal side may diffuse to the circular muscle layer in the intact colon. Treatment of normal sigmoid circular muscle cells for 2 h with undernatants collected from the UC submucosal side (UCS) significantly decreased contraction induced by NKA and thapsigargin and the NKA- and caffeine-induced Ca2+ signal in Ca2+-free medium. In addition, UC mucosa released into the undernatant on its submucosal side significantly more H2O2, IL-1{beta}, and NO than normal mucosa. The reduction in contraction and Ca2+ signal induced by UCS was partially reversed by pretreatment with an IL-1{beta} antibody or with catalase. The NO scavenger hemoglobin partially prevented UCS-induced reduction in contraction and Ca2+ signaling in response to NKA but not the reduced response to thapsigargin or caffeine. Sodium nitroprusside inhibited NKA but not the caffeine-induced Ca2+ signal. We conclude that in UC the mucosa releases IL-1{beta}, H2O2, and NO, which may contribute to the impaired Ca2+ release and altered sigmoid muscle contractility.

neurokinin A; calcium; human; colon


ULCERATIVE COLITIS (UC) is a chronic inflammatory condition that affects the large bowel and most commonly the rectosigmoid area (20). The major symptoms of UC are diarrhea, constipation, and crampy abdominal pain, which may be related to smooth muscle dysfunction. The pathogenesis of UC is not fully understood, but it may depend on inappropriate activation of the mucosal immune system initiated by normal luminal flora or its byproducts (37). Genetic factors confer susceptibility to the development of the disease, and proinflammatory cytokines such as TNF and IL-1{beta} mediate the inflammatory process that eventually leads to clinical manifestations (36).

IL-1{beta} is markedly elevated in colonic mucosa from patients with UC (11, 19), tissue levels of this proinflammatory cytokine correlate with disease activity, and the ratio of the endogenous IL-1 receptor antagonist to IL-1 has shown a close correlation with inflammation (7, 32). Levels of H2O2 are also elevated in UC mucosa and in experimental models of inflammation (14, 21, 43), and local administration of catalase, a H2O2 scavenger, improved colonic mucosal inflammation in a rat model (2, 52). Taken together, these data suggest that IL-1{beta} and H2O2 may play an important role in the pathogenesis of UC.

Abnormal motor function is characteristically found in patients with UC (1, 5, 6, 24, 45, 46, 49) as well as in animal models of colitis (8, 13, 18, 29, 41, 42, 47). Disturbances of normal motor function may affect the defense of the gastrointestinal tract against noxious stimuli present in the lumen. For instance, motor dysfunction in the small intestine (48) and opiate-induced suppression of migrating myoelectrical activity caused bacterial overgrowth, which disappeared once the normal motor pattern was restored (40). Thus disruption of normal motor function in UC may lead to inappropriate growth of enteric flora, which might worsen the inflammation.

We have previously shown that IL-1{beta} and H2O2 may contribute to the sigmoid motor dysfunction of UC (5, 6) because 1) these two mediators are elevated in the muscle layer of affected mucosa; 2) in UC sigmoid circular muscle, the H2O2 scavenger catalase restores the decreased Ca2+ signal and muscle cell shortening in response to the excitatory neurotransmitter neurokinin A (NKA) (4); and 3) IL-1{beta} mimics some of the motor abnormalities observed in UC, such as impaired Ca2+ release mechanisms and reduced muscle contractility in sigmoid circular muscle.

In active UC, increased production of nitric oxide (NO) has been demonstrated in the mucosa (33), and inducible NO synthase activity is significantly increased in colonic mucosa of patients with UC (16, 25, 27, 38). NO is an inhibitory neurotransmitter that mediates smooth muscle relaxation, and high local concentrations of NO may be an important pathogenic step in the development of toxic megacolon (30). Therefore, NO may also contribute to motor dysfunction in patients with UC.

How mucosal inflammation affects colonic motor function in UC and causes increased levels of IL-1{beta} and H2O2 in sigmoid circular muscle is not fully understood. It is possible that inflammatory mediators produced in the UC mucosa and submucosa may directly diffuse to the muscle layer and alter colonic muscle contractility. Therefore, we developed a novel experimental system (Fig. 1) to examine whether the undernatant collected from the submucosal side of UC mucosa and submucosa affects sigmoid smooth muscle contractility and intracellular Ca2+ stores. In this article, we use the term "mucosa" to denote the whole of the mucosa and the submucosa, "UCS" to indicate the undernatant collected from the submucosal side of UC mucosa, and "NS" to indicate the undernatant collected from the submucosal side of normal mucosa. Because inflammatory mediators released from the submucosal side diffuse to the circular muscle, whereas those released from the epithelial side are secreted into the lumen, we have focused on the undernatant collected from the submucosal side. This in vitro model may help to increase the understanding of how the inflammatory mediators produced in the mucosa affect muscle function in patients with UC.



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Fig. 1. After the mucosa and the muscle layer were separated under a microscope, the mucosa was sealed between two tubes, with the luminal side of mucosa facing upward. The Krebs solution on either side of the mucosa was oxygenated with 95% O2-5% CO2. The wall of the bigger tube was perforated to ensure free flow between the tube and the beaker. Histopathological examination of the mucosa after the experiments revealed no equipment-induced damage (data not shown).

 
These data were presented in part at the 106th annual meeting of the American Gastroenterological Association, Chicago, IL, in May 2005.


    METHODS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Tissue specimens. Normal sigmoid colon was obtained from histologically normal margins from cancer resections (n = 12). Inflamed sigmoid colons were obtained from patients with UC undergoing proctocolectomy for active disease refractory to medical treatment (n = 9). Fresh specimens were brought to the laboratory in oxygenated, chilled Krebs solution containing (in mM) 116.6 NaCl, 21.9 NaHCO3, 1.2 KH2PO4, 5.4 dextrose, 1.2 MgCl2, 3.4 KCl, and 2.5 CaCl2. None of control patients had any previous history of colonic motility disorder or evidence of diverticular disease. The experimental protocols were approved by the Human Research Institutional Review Committee at Rhode Island Hospital.

Preparation of mucosa. After removal of the muscle layer and serosa, normal or UC tissue containing mucosa and submucosa was sealed between two tubes as shown in Fig. 1, with the luminal side of the mucosa facing upward in the smaller tube. The luminal side of the mucosa was overlaid with Krebs solution, and the whole setup was maintained at 37°C for 2 h. The Krebs solution on the submucosal side was contained in a bigger tube with holes placed inside a beaker and was collected at the end of the incubation period to measure IL-1{beta}, H2O2, and NO and to treat normal muscle cells. The smaller and bigger tubes were chosen to ensure a good seal. The effectiveness of the seal was ensured by monitoring the level of the solution in the smaller tube. At the end of the experiment, the mucosa was resected along the inner circumference of the smaller tube and used to normalize data per milligram of protein. Histopathological examination of the mucosal tissue after the experiments revealed no equipment-induced damage.

Isolation of smooth muscle cells. After the mucosa and longitudinal muscle layer with serosa were removed by sharp dissection under a microscope, sigmoid circular smooth muscle was cut into small strips (~1 mm wide) and then isolated using enzymatic digestion in HEPES-buffered collagenase solution as described previously (4, 44). Briefly, the collagenase solution (pH 7.2) contained 0.5 mg/ml collagenase Sigma type F, 1 mg/ml papain, 1 mg/ml bovine serum albumin, 1 mM CaCl2, 0.25 mM EDTA, 10 mM glucose, 10 mM HEPES (sodium salt), 4 mM KCl, 125 mM NaCl, 1 mM MgCl2, and 10 mM taurine. The tissue was kept in enzyme solution at 4°C for ~16 h, warmed at room temperature for 30 min, and incubated in a water bath at 31°C for ~30 min. At the end of the digestion period, the tissue was poured over a 200-µm Nitex mesh (Tetko, Elmsford, NY), rinsed in collagenase-free HEPES-buffered solution to remove any trace of collagenase, incubated in this solution at 31°C, and gassed with 100% O2. Collagenase-free HEPES-buffered solution (pH 7.4) contained (in mM) 112.5 NaCl, 3.1 KCl, 2.0 KH2PO4, 10.8 glucose, 24.0 HEPES (sodium salt), 1.9 CaCl2, 0.6 MgCl2, 0.3 mg/ml basal medium Eagle (BME) amino acid supplement, and 0.08 mg/ml soybean trypsin inhibitor. Gentle agitation was used to release single cells.

Agonist-induced contraction of isolated muscle cells. Cells were incubated with Krebs solution (control) or undernatants collected from the beakers containing UCS or NS as described above. The same ratio of protein content in the mucosal tissue per volume of solution in the beaker was used between the NS and UCS mucosa groups. The percentage of viable cells examined using Trypan blue staining was not different between control and UCS-treated cells. When the H2O2 scavenger catalase, an IL-1{beta} antibody, or the NO scavenger hemoglobin were used, the undernatants were preincubated with 78 U/ml catalase, 2 µg/ml IL-1{beta} antibody, 40 µg/ml hemoglobin, or buffer (control) for 30 min and then were used to treat cells. In the cell suspension catalase, IL-1{beta} antibody or hemoglobin was also added. The doses of catalase, IL-1{beta} antibody, and hemoglobin used in the studies were determined in pilot experiments, and the maximally effective dose was used. After cells were incubated with the undernatant or Krebs solution for 2 h, cells were exposed to NKA (10–13 to 10–9 M) for 30 s or to thapsigargin (3 µM) for 15 or 30 s or for 1, 5, 10, or 20 min. Cells exposed to HEPES-buffered solution with vehicle for the same time period as the thapsigargin group were used as the control.

After exposure to NKA or thapsigargin, the cells were fixed in acrolein at a 1% final concentration and refrigerated. For cell length measurement, a drop of the cell-containing medium was placed on a glass slide and 30 consecutive cells from each slide were observed through a phase-contrast microscope (Zeiss, Oberkochen, Germany) and a closed-circuit television camera (model WV-CD51; Panasonic, Secaucus, NJ) connected to a Macintosh computer (Apple, Cupertino, CA). An image software program (National Institutes of Health, Bethesda, MD) was used to acquire images and measure cell length. The average length of 30 cells measured in the absence of agonists was assumed as the control length and compared with lengths measured after the addition of test agents. Shortening was defined as percentage decrease in average length after agonists compared with the control length.

Cytosolic Ca2+ measurements. After cells were incubated with the undernatant or Krebs solution for 2 h, cells were loaded with 1.25 µM fura-2 AM for 30 min and placed into a 5-ml chamber mounted on the stage of an inverted microscope (Zeiss). The cells were allowed to settle onto a coverslip at the bottom of the chamber. NKA (1 µM) or 20 mM caffeine was applied directly to the cells using a pressure ejection micropipette system. Caffeine directly releases Ca2+ from intracellular stores through activation of ryanodine-sensitive channels (12, 34). In our study model, 1 µM NKA was a maximally effective dose because 1 µM NKA and 10–5 M NKA caused the same cell shortening and peak Ca2+ increase (data not shown). Because the tip of the glass pipette was small, only a small volume of solution was ejected from the pipette and reached the cells through the bathing solution. Thus the concentration of the agonists reaching the cells was much lower than that in the micropipette. For instance, to cause maximal cell shortening, 10–9 M NKA was used for cell suspensions and 1 µM NKA was used in a puffing pipette. The concentrations of NKA in these two preparations were 1,000 times different (5). The bathing solution was the same as the collagenase-free HEPES-buffered solution. The Ca2+-free medium was the HEPES-buffered solution without CaCl2, but with 200 µM BAPTA, which completely blocked KCl-induced Ca2+ influx (5). When the Ca2+-free medium was used, the bathing solution was changed twice with Ca2+-free medium after the cells had settled to the bottom of the chamber. The solutions in the pressure ejection micropipettes were identical to the bathing solutions, except for the agonists.

Ca2+ measurements were obtained using a modified dual-excitation wavelength imaging system (IonOptix, Milton, MA) as described previously (4, 5). Ratiometric images were masked in the region outside the borders of the cell because low photon counts produce unreliable ratios near the edges. We developed a method for generating an adaptive mask that follows the borders of the cell as the Ca2+ level changes and as the cell contracts. A pseudoisobestic image (i.e., an image insensitive to Ca2+ concentration changes) was formed in computer memory from a weighted sum of the images generated by 340-nm excitation and 380-nm excitation. This image was then thresholded; that is, values below a selected level were considered to be outside the cell and were assigned a value of 0. For each ratiometric image, the outline of the cell was determined and the generated mask was applied to the ratiometric image. This method allows the simultaneous imaging of the changes in Ca2+ and in cell length. Our algorithm has been incorporated into the IonOptix software. This algorithm calculates the conversion of the fluorescence ratios elicited by 340-nm excitation to 380-nm excitation to Ca2+ concentrations using techniques previously described in detail by Grynkiewicz (15). Peak Ca2+ increase was defined as the difference between the peak value and the basal value.

H2O2 measurement. H2O2 content was measured using the Bioxytech H2O2-560 quantitative hydrogen peroxide assay kit (Oxis International, Portland, OR). This assay is based on the oxidation of ferrous ions (Fe2+) to ferric ions (Fe3+) by H2O2 under acidic conditions. The ferric ion binds with the indicator dye xylenol orange {3,3'-bis[N,N-di(carboxymethyl)-aminomethyl]-o-cresoisulfone-phthalein sodium salt} to form a stable colored complex that can be measured at 560 nm. All measurements were standardized to protein content in the mucosal tissue.

IL-1{beta} measurement. IL-1{beta} concentration was quantified using an IL-1{beta} enzyme immunoassay kit (Cayman Chemical, Ann Arbor, MI) according to the manufacturer's instructions and standardized to protein content in mucosal tissue.

Total nitrite/nitrate measurement. NO is unstable and has a short half-life. The final products of NO in vivo are nitrite (NO2) and nitrate (NO3). Thus total NO production may be estimated by measurement of the sum of NO2 and NO3. Total NO2 and NO3 were measured using a NO3/NO2 colorimetric assay kit (Cayman Chemical) according to the manufacturer's instructions and standardized to protein content.

Protein measurement. The amount of protein was determined using colorimetric analysis (protein assay kit; Bio-Rad Laboratories, Hercules, CA) according to the method of Bradford (3).

Drugs and chemicals. Fura-2 AM and BAPTA were purchased from Molecular Probes (Eugene, OR), thapsigargin was obtained from Calbiochem (San Diego, CA), and IL-1{beta} antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). NKA, H2O2, collagenase type F, papain, catalase, BME amino acid supplement, Na+-HEPES, and other reagents were purchased from Sigma (St. Louis, MO).

Statistical analysis. Data are expressed as means ± SE. Statistical differences between two groups were determined using Student's t-test. Differences between multiple groups were tested using ANOVA and checked for significance using Fisher's protected least- significant difference test.


    RESULTS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Effect of UC undernatant on normal muscle contractility. Consistent with our previous findings, NKA caused a dose-dependent cell shortening of normal sigmoid circular smooth cells in Ca2+-free medium with 200 µM BAPTA. This Ca2+-free medium was used to exclude the effect of Ca2+ influx. In normal sigmoid circular muscle cells pretreated with UCS, NKA-induced contraction was significantly decreased in Ca2+-free medium (n = 6; P < 0.0001, ANOVA) (Fig. 2A), whereas NS had no effect (n = 3) (Fig. 2A), indicating that mediators released from UC mucosa may affect sigmoid circular smooth muscle contraction. Because NKA-induced contraction is mediated by Ca2+ release from intracellular stores (4), these results also suggest that the undernatant of UC mucosa may impair the functional integrity of intracellular Ca2+ stores. In normal sigmoid circular muscle cells pretreated with UCS, contraction in response to diacylglycerol, an activator of PKC, was not affected (Fig. 2C), excluding the possibility that inflammatory mediators from UC mucosa may cause nonspecific damage to the smooth muscle cells.



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Fig. 2. A: neurokinin A (NKA) caused a dose-dependent cell shortening of normal sigmoid circular smooth cells in Ca2+-free medium with 200 µM BAPTA. This Ca2+-free medium blocks KCl-induced contraction (5), demonstrating that in this medium, Ca2+ influx is blocked. In normal sigmoid circular muscle cells pretreated with the undernatant collected from the UC submucosal side (UCS), NKA-induced contraction was significantly decreased in Ca2+-free medium (P < 0.0001; ANOVA), whereas the undernatant collected from the submucosal side of normal mucosa (NS) had no effect, suggesting that inflammatory mediators released from the mucosa in patients with UC may affect sigmoid circular smooth muscle contraction. n = 6 experiments for the control and UCS groups and 3 experiments for the NS group. In each experiment, 30 cells were used for each data point. B: pretreatment of normal cells with UCS significantly reduced thapsigargin-induced contraction (P < 0.0001; ANOVA), whereas NS had no effect. Thapsigargin is an inhibitor of Ca2+-ATPase and inhibits uptake of Ca2+ into stores, causing a net release of Ca2+ and contraction. The data suggest that UCS may affect the integrity of intracellular Ca2+ stores. n = 6 experiments for the control and UCS group and 3 experiments for the NS group. In each experiment, 30 cells were used for each data point. C: in normal sigmoid circular muscle cells pretreated with UCS, contraction in response to diacylglycerol (DAG), an activator of PKC, was not affected in normal Ca2+ medium, excluding the possibility that inflammatory mediators from UC mucosa nonspecifically killed the smooth muscle cells. n = 3 experiments for both groups.

 
Effect of UC undernatant on Ca2+ release from intracellular stores. To further examine whether UCS affects the functional integrity of intracellular Ca2+ stores, we examined thapsigargin-induced contraction. Thapsigargin is an inhibitor of Ca2+-ATPase. It blocks uptake of Ca2+ into stores, causing a net release of Ca2+ and contraction. Pretreatment of normal cells with UCS significantly reduced thapsigargin-induced contraction (n = 6; P < 0.0001, ANOVA), whereas NS had no effect (n = 3) (Fig. 2B).

In addition, we measured NKA- and caffeine-induced increase of intracellular Ca2+ in fura-2 AM-loaded cells. In normal sigmoid circular muscle cells (control group), NKA caused a 356.7 ± 31.1 nM peak Ca2+ increase (from 58.3 ± 4.8 to 415.1 ± 32 nM; 17 cells from 4 patients) in Ca2+-free medium. In cells pretreated with UCS, NKA caused a 140.7 ± 37.7 nM peak Ca2+ increase (from 62.3 ± 7.8 to 203 ± 38.4 nM; 15 cells from 3 patients) in Ca2+-free medium. This increase was significantly lower than that in the control group (P < 0.001; ANOVA) (Fig. 3A).



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Fig. 3. A: in Ca2+-free medium with 200 µM BAPTA, NKA-induced Ca2+ signal was significantly reduced in normal sigmoid muscle cells pretreated with UCS (n = 15 cells from 3 patients; P < 0.001, ANOVA) compared with the control group (n = 17 cells from 4 patients). Pretreatment of normal muscle cells with NS had no effect on NKA-induced Ca2+ signal (n = 13 cells from 3 patients). B: caffeine-induced Ca2+ signal was significantly reduced in normal sigmoid muscle cells pretreated with UCS (n = 17 cells from 3 patients; P < 0.0001, ANOVA) compared with the control group (n = 16 cells from 3 patients). Pretreatment of normal muscle cells with NS had no effect on caffeine-induced Ca2+ signal (n = 12 cells from 3 patients). The data suggest that the undernatant collected from the submucosal side of UC mucosa may impair the mechanisms of Ca2+ release from the intracellular stores.

 
When cells were treated with NS, the Ca2+ signal was the same as that in untreated cells. In cells pretreated with NS, NKA caused a 355.1 ± 44.3 nM peak Ca2+ increase (from 63.1 ± 5.3 to 418.2 ± 45.9 nM; 13 cells from 3 patients) in Ca2+-free medium, an increase that was not different from the control group (Fig. 3A). The data suggest that UCS contained inhibitory mediators that affect NKA-induced Ca2+ release from intracellular stores.

Similarly, in normal sigmoid circular muscle cells (control group), caffeine caused a 387.9 ± 39 nM peak Ca2+ increase (from 61.3 ± 4.1 to 449.2 ± 40.2 nM; 16 cells from 3 patients) in Ca2+-free medium. In cells pretreated with UCS, caffeine caused a 99.6 ± 13.9 nM peak Ca2+ increase (from 57.7 ± 4.8 to 157.3 ± 16.9 nM; 17 cells from 3 patients) in Ca2+-free medium, an increase that was significantly lower than that of the control group (P < 0.0001; ANOVA) (Fig. 3B). In cells pretreated with NS, caffeine caused a 349.4 ± 58.9 nM peak Ca2+ increase (from 56.2 ± 4.6 to 405.6 ± 60.2 nM; 12 cells from 3 patients) in Ca2+-free medium, an increase that was not different from the control group (Fig. 3B). These results confirm that inflammatory mediators released from UCS may impair Ca2+ release mechanisms in sigmoid circular muscle cells.

IL-1{beta}, H2O2, and NO released from the submucosal side of UC mucosa. Our data suggest that inflammatory mediators released from the submucosal side of UC mucosa may affect NKA- and caffeine-induced contraction and/or Ca2+ release from intracellular stores. Figure 4 shows that the levels of total NO2/NO3 were significantly increased in the sigmoid circular muscle tissues of patients with UC compared with normal muscle, suggesting that NO, a relaxant of smooth muscle, may contribute to motor dysfunction in patients with UC. Therefore, we measured IL-1{beta}, H2O2, and NO in the supernatants and undernatants collected from luminal and submucosal sides of normal and UC mucosa. IL-1{beta} (Fig. 5A), H2O2 (Fig. 5B), and total NO2/NO3 (Fig. 5C) were significantly higher in undernatants collected from the submucosal side of UC mucosa than in those from the submucosal side of normal mucosa, suggesting that IL-1{beta}, H2O2, and NO produced in the UC mucosa may directly diffuse to the muscle layer.



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Fig. 4. Levels of total nitrite (NO2)/nitrate (NO3), final products of nitric oxide (NO) in vivo, were significantly increased in UC sigmoid circular muscle (n = 6) compared with normal muscle (n = 6), suggesting that NO production may be increased in UC sigmoid circular muscle (n = 6; *P < 0.05, unpaired t-test).

 


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Fig. 5. Levels of IL-1{beta} (A) and H2O2 (B) as well as the final products of NO (C) were significantly higher in the undernatants collected from UCS than in the undernatants collected from NS, suggesting that IL-1{beta}, H2O2, and NO produced in the UC mucosa may directly diffuse to the muscle layer. Levels of IL-1{beta} (A), H2O2 (B), and final products of NO (C) were also significantly higher in the supernatants collected from the luminal side of UC mucosa than in the supernatants collected from the luminal side of normal mucosa. *P < 0.05 and **P < 0.001 (unpaired t-test) compared with normal mucosa group. Note that we use the term "mucosa" to refer to the whole of the mucosa and the submucosa.

 
IL-1{beta} (Fig. 5A) and total NO2/NO3 (Fig. 5C) were also significantly higher in the supernatants collected from the luminal side of UC mucosa than in normal mucosa. H2O2 in the supernatants from the luminal side of normal and UC mucosa was not measurable.

To test whether these three mediators present in the undernatants of UC mucosa affect sigmoid muscle contractility, we pretreated the undernatant with 2 µg/ml IL-1{beta} antibody, 80 U/ml H2O2 scavenger catalase, and 40 µg/ml NO scavenger hemoglobin for 30 min. Pretreatment with IL-1{beta} antibody significantly prevented UCS-induced reduction of muscle contraction in response to NKA and thapsigargin (Figs. 6A and 7A). Pretreatment with IL-1{beta} antibody also partially prevented UCS-induced reduction of peak Ca2+ increase in response to NKA and caffeine in Ca2+-free medium (Figs. 8A and 9A). Similarly, pretreatment with catalase prevented UCS-induced reduction of muscle contraction in response to NKA and thapsigargin (Figs. 6A and 7A) as well as UCS-induced reduction of peak Ca2+ increase in response to NKA and caffeine in Ca2+-free medium (Figs. 8A and 9A).



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Fig. 6. UCS and NS were pretreated with 2 µg/ml IL-1{beta} antibody, 80 U/ml H2O2 scavenger catalase, and 40 µg/ml hemoglobin for 30 min and then were used to treat normal sigmoid circular muscle cells for 2 h. A: in Ca2+-free medium with 200 µM BAPTA, pretreatment with IL-1{beta} antibody, catalase, or hemoglobin significantly prevented UCS-induced reduction of muscle contraction in response to NKA (P < 0.0001, P < 0.0001, and P < 0.05, respectively; ANOVA). B: pretreatment of NS with IL-1{beta} antibody, catalase, or hemoglobin had no effect on NKA-induced contraction. These data suggest that IL-1{beta}, H2O2, and NO may be released from UC mucosa and affect sigmoid muscle contractility. n = 6 experiments for control and UCS groups and 3 experiments for other groups. In each experiment, 30 cells were used for each data point.

 


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Fig. 7. UCS and NS were pretreated with 2 µg/ml IL-1{beta} antibody, 80 U/ml H2O2 scavenger catalase, and 40 µg/ml hemoglobin for 30 min and then were used to treat normal sigmoid circular muscle cells for 2 h. A: pretreatment with IL-1{beta} antibody or catalase significantly prevented UCS-induced reduction of muscle contraction in response to thapsigargin (P < 0.0001; ANOVA), whereas hemoglobin had no effect. B: pretreatment of NS with IL-1{beta} antibody, catalase, or hemoglobin had no effect on thapsigargin-induced contraction. These data suggest that IL-1{beta} and H2O2 released from UC mucosa may affect the integrity of intracellular Ca2+ stores of sigmoid circular muscle but that NO may not. n = 6 experiments for control and UCS groups and 3 experiments for other groups. In each experiment, 30 cells were used for each data point.

 


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Fig. 8. A: in Ca2+-free medium with 200 µM BAPTA, pretreatment with IL-1{beta} antibody, catalase, or hemoglobin (Hb) significantly prevented UCS-induced reduction of peak Ca2+ increase in response to NKA. B: pretreatment of NS with IL-1{beta} antibody, catalase, or hemoglobin had no effect on NKA-induced peak Ca2+ increase. These data suggest that IL-1{beta}, H2O2, and NO may be released from UC mucosa and affect the mechanisms of NKA-induced Ca2+ release from intracellular stores in UC sigmoid circular muscle. n = ~3–6 patients in each group, and the total number of cells used in each group is shown under the bars. **P < 0.0001 (ANOVA) compared with control. *P < 0.05 (ANOVA) compared with UCS group. {clubsuit}P < 0.001 (ANOVA) compared with UCS group.

 


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Fig. 9. A: in Ca2+-free medium with 200 µM BAPTA, pretreatment with IL-1{beta} antibody or catalase significantly prevented UCS-induced reduction of peak Ca2+ increase in response to caffeine, whereas hemoglobin had no effect. B: pretreatment of NS with IL-1{beta} antibody, catalase, or hemoglobin had no effect on caffeine-induced peak Ca2+ increase. These data suggest that IL-1{beta} and H2O2 in UCS, but not in NO, may affect mechanisms of caffeine-induced Ca2+ release from intracellular stores of sigmoid circular muscle. n = 3~6 patients each group, and the total number of cells used in each group is shown under the bars. **P < 0.0001 (ANOVA) compared with control. *P < 0.001 (ANOVA) compared with UCS group.

 
Hemoglobin was less effective than IL-1{beta} antibodies or catalase in restoring the amplitude of contraction or Ca2+ signal in response to agonists, although the effect of hemoglobin on NKA-induced muscle contraction and Ca2+ peak was statistically significant (Figs. 6A and 8A). Hemoglobin, however, did not affect UCS-induced reduction of muscle contraction in response to thapsigargin (Fig. 7A) and UCS-induced reduction of Ca2+ peak in response to caffeine (Fig. 9A).

Treatment of normal muscle cells with sodium nitroprusside (10–5 M), a donor of NO, significantly reduced the NKA-induced Ca2+ increase in Ca2+-free medium (Fig. 10A) but did not affect the caffeine-induced Ca2+ increase (Fig. 10B). The data indicate that NO may impair NKA-induced Ca2+ release from intracellular stores but may not affect the mechanisms of caffeine-induced Ca2+ release.



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Fig. 10. In Ca2+-free medium with 200 µM BAPTA, treatment of normal muscle cells with sodium nitroprusside (SNP; 10–5M), a donor of NO, significantly reduced NKA-induced peak Ca2+ increase (A) but did not affect caffeine-induced peak Ca2+ increase (B). Data indicate that NO may impair mechanisms of NKA- but not caffeine-induced Ca2+ release from intracellular stores. n = 3 patients each group, and the total number of cells used in each group is shown under the bars. **P < 0.0001, unpaired t-test.

 
Pretreatment of NS with IL-1{beta} antibody, catalase, and hemoglobin had no effect on agonist-induced contraction and peak Ca2+ increase (Figs. 6B, 7B, 8B, and 9B).

These results suggest that IL-1{beta}, H2O2, and NO are released from UC mucosa, diffuse to the muscle layer, and affect motor function in the sigmoid colon in patients with UC.


    DISCUSSION
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We have previously shown that in the normal sigmoid colonic circular muscle, NKA may be physiologically important as an excitatory neurotransmitter. NKA activates neurokinin 2 (NK2) receptors linked to a calmodulin-dependent signal transduction pathway, requiring the release of intracellular Ca2+ (4). In addition, the NKA-activated NK2 receptors are linked to a PKC-dependent contractile pathway (6).

In the sigmoid colonic circular muscle layer in patients with UC, however, contraction in response to NKA and to electrical field stimulation is reduced (6, 49), while the NKA-induced Ca2+ release is decreased (5), suggesting an impairment of mechanisms of Ca2+ release from intracellular stores. In addition, in UC circular muscle, NKA-induced contraction is blocked by PKC inhibitors but not by calmodulin inhibitors, indicating a change in the contractile signal transduction pathway from calmodulin and PKC dependent in normal cells to only PKC dependent in UC (6). We also found that levels of H2O2 and IL-1{beta} are significantly increased in human sigmoid circular muscle from patients with UC. The H2O2 scavenger catalase restored the decreased Ca2+ signal in response to NKA in UC sigmoid circular muscle and restored cell shortening to almost normal levels, suggesting that H2O2 contributes to sigmoid motor dysfunction in UC. In addition, IL-1{beta} mimics these changes observed in UC, suggesting that IL-1{beta} may also contribute to sigmoid motor dysfunction in UC. In the present study, we also found that NO, a relaxant of smooth muscle, was significantly increased in UC sigmoid muscle (Fig. 4). Together, these data suggest that the inflammatory mediators present in sigmoid circular muscle may affect muscle function in patients with UC.

It is known that IL-1{beta} (11, 19), H2O2 (14, 21, 43), and NO (33) are elevated in the colonic mucosa of patients with UC and that IL-1{beta} and H2O2 are released into culture medium from mucosal explants (10). Whether these inflammatory mediators produced in the UC mucosa diffuse to the muscle layer and affect muscle contractility is not known.

In the present study, we assembled a new experimental setup (Fig. 1) to study whether factors derived from the submucosal side of UC mucosa affect normal smooth muscle contraction. We found that treatment of normal sigmoid muscle cells for 2 h with UCS significantly decreased NKA- and thapsigargin-induced contraction (Fig. 2) as well as the NKA- and caffeine-induced Ca2+ signal in Ca2+-free medium with 200 µM BAPTA (Fig. 3). We have previously shown that this Ca2+-free medium to effectively block KCl-induced intracellular Ca2+ increase (5) without affecting NKA-induced contraction and intracellular Ca2+ increase, demonstrating that Ca2+ influx is selectively blocked by incubation in this Ca2+-free medium.

It is known that the NKA- and caffeine-induced Ca2+ signal is mediated by release from intracellular Ca2+ stores. Thapsigargin is a naturally occurring sesquiterpene lactone isolated from the umbelliferous plant Thapsia garganica. It inhibits sarcoplasmic reticulum Ca2+-ATPase (9), thus inhibiting the uptake of Ca2+ into stores and causing a net release of Ca2+ and contraction. Caffeine directly releases Ca2+ from intracellular stores through activation of ryanodine-sensitive channels (12, 34). Because UC undernatants block contraction and/or intracellular Ca2+ increase in response to thapsigargin, NKA, and caffeine, our data suggest that the undernatant contains factors impairing the mechanisms of Ca2+ release from intracellular stores. In particular, our data suggest that H2O2, IL-1{beta}, and NO produced in UC mucosa and/or submucosa affect circular muscle contractility and Ca2+ release from intracellular stores because 1) UC mucosa released significantly more H2O2, IL-1{beta}, and NO into the submucosal side than normal mucosa (Fig. 5), and 2) UCS-induced reduction in contraction and Ca2+ signal in response to NKA was partially prevented by pretreatment with the H2O2 scavenger catalase, an IL-1{beta} antibody, or the NO scavenger hemoglobin (Figs. 6A and 8A).

Pretreatment of UCS with catalase or with an IL-1{beta} antibody significantly prevented UCS-induced reduction in contraction in response to thapsigargin (Fig. 7A) and reduction in Ca2+ signal in response to caffeine (Fig. 9A). However, pretreatment with hemoglobin did not prevent UCS-induced reduction in contraction and Ca2+ signal in response to thapsigargin (Fig. 7A) and caffeine (Fig. 9A), respectively. In addition, sodium nitroprusside, a donor of NO, significantly reduced NKA-induced peak Ca2+ increase in Ca2+-free medium (Fig. 10A) but did not affect the caffeine-induced peak Ca2+ increase in Ca2+-free medium (Fig. 10B). These data suggest that IL-1{beta} and H2O2 may impair mechanisms of the NKA- and caffeine-induced Ca2+ release, whereas NO affects mechanisms of NKA- but not caffeine-induced Ca2+ release from intracellular stores. The inhibitory effect of NO appears to be mediated by increasing intracellular cGMP and by activating cGMP-dependent protein kinases (PKG) (17, 35, 50). It has been reported that PKG can directly phosphorylate PLC-{beta}3 and PLC-{beta}2 and then decrease the G protein-activated inositol 1,4,5-trisphosphate (IP3) release as a result of blocking PLC-{beta} activation (51). PKG can also phosphorylate IP3 receptor type I and inhibit IP3-dependent Ca2+ release in gastric smooth muscle (31). It is possible that overproduction of NO in UC may activate these cGMP-dependent pathways to inhibit NKA-induced Ca2+ release and contraction.

Pretreatment of NS with the IL-1{beta} antibody, catalase, or hemoglobin did not affect agonist-induced contraction and peak Ca2+ increase (Figs. 6B, 7B, 8B, and 9B), demonstrating that IL-1{beta} antibody, catalase, and hemoglobin by themselves have no effect on muscle contraction and Ca2+ signal as previously shown (5).

Catalase almost completely prevented UCS-induced reduction of muscle contraction and Ca2+ signal, suggesting that H2O2 may be an important mediator causing motor dysfunction in patients with UC. This is consistent with previous findings (5). IL-1{beta} antibody only partially prevented the effect of UCS, suggesting that other inflammatory mediators such as H2O2 are still present to affect muscle contractility after the IL-1{beta} antibody neutralized IL-1{beta} in the undernatant. We have previously shown that the IL-1{beta}-induced reduction in Ca2+ signal and the shortening of enzymatically isolated circular muscle cells in response to NKA are partially restored by catalase and that 2-h treatment of sigmoid circular muscle with IL-1{beta} significantly increases H2O2 production (6), suggesting that the effect of IL-1{beta} is mediated at least in part by the production of H2O2. Similarly to IL-1{beta} antibody, hemoglobin partially prevented the effect of UCS, suggesting that other inflammatory mediators, such as IL-1{beta} and H2O2, are still present to affect muscle contractility after the removal of NO in the cell suspension. H2O2 has been reported to cause production of NO in neurons (28) and endothelial cells (26). Therefore, it is possible that H2O2 present in UCS might cause the production of NO in sigmoid muscle cells. Removal of H2O2 by catalase might inhibit H2O2-induced NO production by sigmoid muscle cells. This may explain why catalase alone almost completely prevented UCS-induced reduction in contraction and Ca2+ signal.

The mediators released from the submucosal side are the same as those found in the circular muscle layer, suggesting that they may originate in the mucosa at least in the initial stage of inflammation. Therefore, it is possible that in the early stage of disease, inflammatory mediators derived from the mucosa may affect muscle function in patients with UC. Later, in addition to being targets of inflammatory mediators, smooth muscle cells may also be a source of these mediators and contribute to the maintenance of inflammation in patients with UC. This possibility is supported by considerable experimental evidence. The expression of IL-1{beta} mRNA is significantly increased in the colonic muscle of rats with acetic acid-induced colitis (22). The activation of the IL-1 type I receptor by IL-1{beta} during inflammation may result in the release of IL-6 (23). In addition, cultured human colonic smooth muscle cells secrete IL-1{beta}, IL-6, and IL-8 after stimulation with a mixture of TNF-{alpha}, IL-1{beta}, and interferon-{gamma} (39). H2O2 production was increased in the muscularis of the inflamed colon in dextran sodium sulfate-treated rats (13). Isolated sigmoid smooth muscle cells from patients with UC contained excess H2O2 compared with normal cells (5).

In conclusion, the data obtained in this novel in vitro mucosa system indicate that in UC, IL-1{beta}, H2O2, and NO are produced in the inflamed mucosa and/or submucosa, diffuse into the adjacent microenvironment, and presumably affect circular muscle function by contributing to the impairment of Ca2+ release and altered sigmoid muscle contractility typically observed in this disease.


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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant R21 DK-62775-01 (to W. Cao).


    FOOTNOTES
 

Address for reprint requests and other correspondence: W. Cao, Dept. of Medicine, Brown Medical School and Rhode Island Hospital, 55 Claverick St., Rm. 337, Providence, RI 02903 (e-mail: Weibiao_Cao{at}brown.edu)

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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